Methods for increasing observed editing in bacteria

ABSTRACT

The present disclosure relates to methods for increasing observed editing rates in the surviving bacteria cells.

RELATED CASES

This utility patent application claims priority to U.S. Ser. No.62/937,289, filed 19 Nov. 2019, entitled “Methods for IncreasingObserved Editing in Bacteria.”

FIELD OF THE INVENTION

The present disclosure relates to methods for increasing observedediting rates in nucleic acid-guided nuclease editing bacteria cells.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underthe applicable statutory provisions.

The ability to make precise, targeted changes to the genome of livingcells has been a long-standing goal in biomedical research anddevelopment. Recently, various nucleases have been identified that allowfor manipulation of gene sequences, and hence gene function. Thesenucleases include nucleic acid-guided nucleases, which enableresearchers to generate permanent edits in live cells. Of course, it isdesirable to decrease the background of unedited cells so that editedcells are more readily identified.

There is thus a need in the art of nucleic acid-guided nuclease editingfor improved methods, compositions, modules and instruments forincreasing the percentage and diversity of edited cells in a cellpopulation post-transformation. The present disclosure addresses thisneed.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter. Other features, details,utilities, and advantages of the claimed subject matter will be apparentfrom the following written Detailed Description including those aspectsillustrated in the accompanying drawings and defined in the appendedclaims.

Thus, presented herein is a method for nuclease-directed nucleaseediting comprising: providing electrocompetent bacteria cells; providingan engine vector comprising a promoter driving expression of a codingsequence for a nucleic acid-guided nuclease; a bacterial origin ofreplication; a promoter driving expression of a coding sequence for arecA protein; and a selection marker; providing an editing vectorcomprising a promoter driving transcription of at least two editingcassettes where each editing cassette comprises a gRNA sequence and adonor DNA sequence to be transcribed; a bacterial origin of replication;and a selection marker; transforming the electrocompetent bacterialcells with the engine and editing vectors; allowing the transformedcells to edit; and pooling the edited cells or selecting small coloniesof edited cells. In some aspects, the nuclease is MAD7 and in otheraspects the nuclease is Cas9. In some aspects, the coding sequence for arecA protein is a coding sequence for a recA fusion protein, and in someaspects, the recA fusion protein is a recA-srpR fusion protein. In someaspects, the method further comprises the steps of, after the pooling orselecting step: making the edited bacterial cells electrocompetent;providing a second editing vector comprising a promoter drivingtranscription of at least two editing cassettes where each editingcassette comprises a gRNA sequence and a donor DNA sequence to betranscribed; a bacterial origin of replication; and a selection marker;transforming the electrocompetent bacterial cells with the secondediting vector; allowing the transformed cells to edit; and pooling thetwice-edited cells. Other embodiments provide modules and integratedinstruments for processing and editing the bacterial cells, as well ascompositions of matter comprising the engine and editing vectors.

These aspects and other features and advantages of the invention aredescribed below in more detail.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments taken in conjunction with the accompanyingdrawings in which:

FIG. 1A is a simple process diagram for editing in bacteria cells. FIG.1B is a vector map for the p197 engine vector. FIG. 1C is a vector mapfor the p197-recA engine vector. FIG. 1D is a vector map for thep197-srpRrecA engine vector. FIG. 1E is a simplified rendering of anediting plasmid having a “3-pack” multiplex or compound editingcassette.

FIGS. 2A-2C depict three different views of an exemplary automatedmulti-module cell processing instrument for performing nucleicacid-guided nuclease editing employing a split nuclease tetheringsystem.

FIG. 3A depicts one embodiment of a rotating growth vial for use withthe cell growth module described herein and in relation to FIGS. 3B-3D.FIG. 3B illustrates a perspective view of one embodiment of a rotatinggrowth vial in a cell growth module housing. FIG. 3C depicts a cut-awayview of the cell growth module from FIG. 3B. FIG. 3D illustrates thecell growth module of FIG. 3B coupled to LED, detector, and temperatureregulating components.

FIG. 4A depicts retentate (top) and permeate (center) members for use ina tangential flow filtration module (e.g., cell growth and/orconcentration module), as well as the retentate and permeate membersassembled into a tangential flow assembly (bottom). FIG. 4B depicts twoside perspective views of a reservoir assembly of a tangential flowfiltration module. FIGS. 4C-4E depict an exemplary top, with fluidic andpneumatic ports and gasket suitable for the reservoir assemblies shownin FIG. 4B.

FIG. 5A depicts an exemplary combination reagent cartridge andelectroporation device (e.g., transformation module) that may be used ina multi-module cell processing instrument. FIG. 5B is a top perspectiveview of one embodiment of an exemplary flow-through electroporationdevice that may be part of a reagent cartridge.

FIG. 5C depicts a bottom perspective view of one embodiment of anexemplary flow-through electroporation device that may be part of areagent cartridge. FIGS. 5D-5F depict a top perspective view, a top viewof a cross section, and a side perspective view of a cross section of anFTEP device useful in a multi-module automated cell processinginstrument such as that shown in FIGS. 2A-2C.

FIGS. 6A-6C depict an embodiment of a solid wall isolation incubationand normalization (SWIIN) module. FIG. 6D depicts the embodiment of theSWIIN module in FIGS. 6A-6C further comprising a heater and a heatedcover.

FIG. 7A shows the CFUs obtained for E coli transformed with the p197engine vector vs. the p197-recA engine vector at uptake, at plating, andpost-plating. FIG. 7B shows the percentage of triple simultaneous editsfor E. coli cells transformed with the p197 engine vector vs. thep197-recA engine vector.

FIG. 8A shows the CFUs obtained for E coli transformed with the p197engine vector, the p197-recA engine vector, and the p197-srpRrecA enginevector at uptake, at plating, and post-plating. FIG. 8B shows theediting CFU and percentage of triple simultaneous edits for E. colicells transformed with the p197 engine vector, the p197-recA enginevector and the p197-srpRrecA engine vector. FIG. 8C is a table showingthe percentage post-plating survival rate and the percentage editingrate for E. coli cells transformed with the p197 engine vector, thep197-recA engine vector and the p19′7-srpRrecA engine vector.

It should be understood that the drawings are not necessarily to scale,and that like reference numbers refer to like features.

DETAILED DESCRIPTION

All of the functionalities described in connection with one embodimentof the methods, devices or instruments described herein are intended tobe applicable to the additional embodiments of the methods, devices andinstruments described herein except where expressly stated or where thefeature or function is incompatible with the additional embodiments. Forexample, where a given feature or function is expressly described inconnection with one embodiment but not expressly mentioned in connectionwith an alternative embodiment, it should be understood that the featureor function may be deployed, utilized, or implemented in connection withthe alternative embodiment unless the feature or function isincompatible with the alternative embodiment.

The practice of the techniques described herein may employ, unlessotherwise indicated, conventional techniques and descriptions ofmolecular biology (including recombinant techniques), cell biology,biochemistry, and genetic engineering technology, which are within theskill of those who practice in the art. Such conventional techniques anddescriptions can be found in standard laboratory manuals such as Greenand Sambrook, Molecular Cloning: A Laboratory Manual. 4th, ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (2014);Current Protocols in Molecular Biology, Ausubel, et al. eds., (2017);Neumann, et al., Electroporation and Electrofusion in Cell Biology,Plenum Press, New York, 1989; and Chang, et al., Guide toElectroporation and Electrofusion, Academic Press, California (1992),all of which are herein incorporated in their entirety by reference forall purposes Nucleic acid-guided nuclease techniques can be found in,e.g., Genome Editing and Engineering from TALENs and CRISPRs toMolecular Surgery, Appasani and Church (2018); and CRISPR: Methods andProtocols, Lindgren and Charpentier (2015); both of which are hereinincorporated in their entirety by reference for all purposes.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a cell” refers toone or more cells, and reference to “the system” includes reference toequivalent steps, methods and devices known to those skilled in the art,and so forth. Additionally, it is to be understood that terms such as“left,” “right,” “top,” “bottom,” “front,” “rear,” “side,” “height,”“length,” “width,” “upper,” “lower,” “interior,” “exterior,” “inner,”“outer” that may be used herein merely describe points of reference anddo not necessarily limit embodiments of the present disclosure to anyparticular orientation or configuration. Furthermore, terms such as“first,” “second,” “third,” etc., merely identify one of a number ofportions, components, steps, operations, functions, and/or points ofreference as disclosed herein, and likewise do not necessarily limitembodiments of the present disclosure to any particular configuration ororientation.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated by reference for the purpose of describing anddisclosing devices, formulations and methodologies that may be used inconnection with the presently described invention.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in smaller ranges, and arealso encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, features and procedures well known to thoseskilled in the art have not been described in order to avoid obscuringthe invention. The terms used herein are intended to have the plain andordinary meaning as understood by those of ordinary skill in the art.

The term “complementary” as used herein refers to Watson-Crick basepairing between nucleotides and specifically refers to nucleotideshydrogen-bonded to one another with thymine or uracil residues linked toadenine residues by two hydrogen bonds and cytosine and guanine residueslinked by three hydrogen bonds. In general, a nucleic acid includes anucleotide sequence described as having a “percent complementarity” or“percent homology” to a specified second nucleotide sequence. Forexample, a nucleotide sequence may have 80%, 90%, or 100%complementarity to a specified second nucleotide sequence, indicatingthat 8 of 10, 9 of 10 or 10 of 10 nucleotides of a sequence arecomplementary to the specified second nucleotide sequence. For instance,the nucleotide sequence 3′-TCGA-5′ is 100% complementary to thenucleotide sequence 5′-AGCT-3′; and the nucleotide sequence 3′-TCGA-5′is 100% complementary to a region of the nucleotide sequence5′-TAGCTG-3′.

The term DNA “control sequences” refers collectively to promotersequences, polyadenylation signals, transcription termination sequences,upstream regulatory domains, origins of replication, internal ribosomeentry sites, nuclear localization sequences, enhancers, and the like,which collectively provide for the replication, transcription andtranslation of a coding sequence in a recipient cell. Not all of thesetypes of control sequences need to be present so long as a selectedcoding sequence is capable of being replicated, transcribed and—for somecomponents—translated in an appropriate host cell.

As used herein the terms “donor DNA” or “donor nucleic acid” and“homology arm” all refer to nucleic acid that is designed to introduce aDNA sequence modification (insertion, deletion, substitution) into alocus (e.g., a target genomic DNA sequence or cellular target sequence)by homologous recombination using nucleic acid-guided nucleases. Forhomology-directed repair, the donor DNA must have sufficient homology tothe regions flanking the “cut site” or site to be edited in the genomictarget sequence. The length of the homology arm(s) will depend on, e.g.,the type and size of the modification being made. In many instances andpreferably, the donor DNA will have two regions of sequence homology(e.g., two homology arms) to the genomic target locus. Preferably, an“insert” region or “DNA sequence modification” region—the nucleic acidmodification that one desires to be introduced into a genome targetlocus in a cell-will be located between two regions of homology. The DNAsequence modification may change one or more bases of the target genomicDNA sequence at one specific site or multiple specific sites. A changemay include changing 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 75,100, 150, 200, 300, 400, or 500 or more base pairs of the genomic targetsequence. A deletion or insertion may be a deletion or insertion of 1,2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 300, 400, or500 or more base pairs of the genomic target sequence.

The terms “guide nucleic acid” or “guide RNA” or “gRNA” refer to apolynucleotide comprising 1) a guide sequence capable of hybridizing toa genomic target locus, and 2) a scaffold sequence capable ofinteracting or complexing with a nucleic acid-guided nuclease. In thecontext of this disclosure, a gRNA is linked to a capture sequence toallow for capture of the gRNA.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or, more often in the context of the presentdisclosure, between two nucleic acid molecules. The term “homologousregion” or “homology arm” refers to a region on the donor DNA with acertain degree of homology with the target genomic DNA sequence.Homology can be determined by comparing a position in each sequencewhich may be aligned for purposes of comparison. When a position in thecompared sequence is occupied by the same base or amino acid, then themolecules are homologous at that position. A degree of homology betweensequences is a function of the number of matching or homologouspositions shared by the sequences.

“Operably linked” refers to an arrangement of elements where thecomponents so described are configured so as to perform their usualfunction. Thus, control sequences operably linked to a coding sequenceare capable of effecting the transcription, and in some cases, thetranslation, of a coding sequence. The control sequences need not becontiguous with the coding sequence so long as they function to directthe expression of the coding sequence. Thus, for example, interveninguntranslated yet transcribed sequences can be present between a promotersequence and the coding sequence and the promoter sequence can still beconsidered “operably linked” to the coding sequence. In fact, suchsequences need not reside on the same contiguous DNA molecule (i.e.chromosome) and may still have interactions resulting in alteredregulation.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably. Proteins may or may not be made up entirely of aminoacids.

A “promoter” or “promoter sequence” is a DNA regulatory region capableof binding RNA polymerase and initiating transcription of apolynucleotide or polypeptide coding sequence such as messenger RNA,ribosomal RNA, small nuclear or nucleolar RNA, guide RNA, or any kind ofRNA transcribed by any class of any RNA polymerase I, II or III.Promoters may be constitutive or inducible.

As used herein the term “selectable marker” or “selection marker” or“survival marker” refers to a gene introduced into a cell that confers atrait suitable for artificial selection. General use selectable markersare well-known to those of ordinary skill in the art and includeampilcillin/carbenicillin, kanamycin, chloramphenicol, nourseothricinN-acetyl transferase, erythromycin, tetracycline, gentamicin, bleomycin,streptomycin, puromycin or other selectable markers that may beemployed.

The term “specifically binds” as used herein includes an interactionbetween two molecules, e.g., a capture sequence (e.g., a poly-dTsequence) and the sequence to be captured, with a binding affinityrepresented by a dissociation constant of about 10⁻⁷ M, about 10⁻⁸ M,about 10⁻⁹ M, about 10⁻¹⁰ M, about 10⁻¹¹ M, about 10⁻¹² M, about 10⁻¹³M, about 10⁻¹⁴M or about 10⁻¹⁵M.

The terms “target genomic DNA sequence”, “cellular target sequence”,“target sequence”, or “genomic target locus” refer to any locus in vitroor in vivo, or in a nucleic acid (e.g., genome or episome) of a cell orpopulation of cells, in which a change of at least one nucleotide isdesired using a nucleic acid-guided nuclease editing system. The targetsequence can be a genomic locus or extrachromosomal locus.

The term “variant” may refer to a polypeptide or polynucleotide thatdiffers from a reference polypeptide or polynucleotide but retainsessential properties. A typical variant of a polypeptide differs inamino acid sequence from another reference polypeptide. Generally,differences are limited so that the sequences of the referencepolypeptide and the variant are closely similar overall and, in manyregions, identical. A variant and reference polypeptide may differ inamino acid sequence by one or more modifications (e.g., substitutions,additions, and/or deletions). A variant of a polypeptide may be aconservatively modified variant. A substituted or inserted amino acidresidue may or may not be one encoded by the genetic code (e.g., anon-natural amino acid). A variant of a polypeptide may be naturallyoccurring, such as an allelic variant, or it may be a variant that isnot known to occur naturally.

A “vector” is any of a variety of nucleic acids that comprise a desiredsequence or sequences to be delivered to and/or expressed in a cell.Vectors are typically composed of DNA, although RNA vectors are alsoavailable. Vectors include, but are not limited to, plasmids, fosmids,phagemids, virus genomes, synthetic chromosomes, and the like. In someembodiments, two vectors are employed comprising 1) an engine vector,comprising the coding sequence for a nucleic acid-guided nuclease and inthe method and composition embodiments herein, the coding sequence forthe recA protein or recA fusion protein; and 2) an editing vector,comprising at least two editing cassettes each comprising a gRNA and adonor DNA sequence. In alternative embodiments, all editing components,including the nucleic acid-guided nuclease, recA protein codingsequence, gRNA, and donor DNA sequence are all on the same vector (e.g.,a combined editing/engine vector).

Nuclease-Directed Genome Editing Generally

The compositions and methods described herein are employed to performnuclease-directed genome editing to introduce at least two simultaneousdesired edits to a population of bacteria cells. In some embodiments,recursive cell editing is performed where edits are introduced insuccessive rounds of editing. A nucleic acid-guided nuclease complexedwith an appropriate synthetic guide nucleic acid (e.g., gRNA) in a cellcan cut the genome of the cell at a desired location. The guide nucleicacid helps the nucleic acid-guided nuclease recognize and cut the DNA ata specific target sequence. By manipulating the nucleotide sequence ofthe guide nucleic acid, the nucleic acid-guided nuclease may beprogrammed to target any DNA sequence for cleavage as long as anappropriate protospacer adjacent motif (PAM) is nearby. Thus, the gRNAcomprises homology to the target sequence and can be used to track theedit made to the target sequence. In certain aspects, the nucleicacid-guided nuclease editing system may use two separate guide nucleicacid molecules that combine to function as a guide nucleic acid, e.g., aCRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). In otheraspects and preferably, the guide nucleic acid is a single guide nucleicacid construct that includes both 1) a guide sequence capable ofhybridizing to a genomic target locus, and 2) a scaffold sequencecapable of interacting or complexing with a nucleic acid-guidednuclease.

In general, a guide nucleic acid (e.g., gRNA) complexes with acompatible nucleic acid-guided nuclease and can then hybridize with atarget sequence, thereby directing the nuclease to the target sequence.A guide nucleic acid can be DNA or RNA; alternatively, a guide nucleicacid may comprise both DNA and RNA. In some embodiments, a guide nucleicacid may comprise modified or non-naturally occurring nucleotides. Incases where the guide nucleic acid comprises RNA, the gRNA may beencoded by a DNA sequence on a polynucleotide molecule such as aplasmid, linear construct, or the coding sequence may and preferablydoes reside within an editing cassette. Methods and compositions fordesigning and synthesizing editing cassettes are described in U.S. Pat.Nos. 10,240,167; 10,266,849; 9,982,278; 10,351,877; 10,364,442;10,435,715; and 10,465,207, all of which are incorporated herein intheir entirety. U.S. Pat. No. 10,465,207 is drawn to multiplex editingcassettes (e.g., two or more editing cassettes targeting differentregions in the genome) as used in the method embodiments employedherein. Editing cassettes, in addition to paired gRNAs and donor DNAs,may and typically do comprise additional sequences such as bar codes.

A guide nucleic acid comprises a guide sequence, where the guidesequence is a polynucleotide sequence having sufficient complementaritywith a target sequence to hybridize with the target sequence and directsequence-specific binding of a complexed nucleic acid-guided nuclease tothe target sequence. Because of this complementarity, the gRNA sequencemay serve as a proxy for the edit made. The degree of complementaritybetween a guide sequence and the corresponding target sequence, whenoptimally aligned using a suitable alignment algorithm, is about or morethan about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.Optimal alignment may be determined with the use of any suitablealgorithm for aligning sequences. In some embodiments, a guide sequenceis about or more than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or morenucleotides in length. In some embodiments, a guide sequence is lessthan about 75, 50, 45, 40, 35, 30, 25, 20 nucleotides in length.Preferably the guide sequence is 10-30 or 15-20 nucleotides long, or 15,16, 17, 18, 19, or 20 nucleotides in length.

In general, to generate an edit in the target sequence the gRNA/nucleasecomplex binds to a target sequence as determined by the guide RNA, andthe nuclease recognizes a protospacer adjacent motif (PAM) sequenceadjacent to the target sequence. The target sequence can be anypolynucleotide endogenous or exogenous to the cell, or in vitro. Forexample, the target sequence can be a polynucleotide residing in thenucleus of the cell. A target sequence can be a sequence encoding a geneproduct (e.g., a protein) or a non-coding sequence (e.g., a regulatorypolynucleotide, an intron, a PAM, a control sequence, or “junk” DNA).

The guide nucleic acid may be and preferably is part of an editingcassette that encodes the donor nucleic acid that targets a cellulartarget sequence. Alternatively, the guide nucleic acid may not be partof the editing cassette and instead may be encoded on the editing vectorbackbone. For example, a sequence coding for a guide nucleic acid can beassembled or inserted into a vector backbone first, followed byinsertion of the donor nucleic acid in, e.g., an editing cassette. Inother cases, the donor nucleic acid in, e.g., an editing cassette can beinserted or assembled into a vector backbone first, followed byinsertion of the sequence coding for the guide nucleic acid. Preferably,the sequence encoding the guide nucleic acid and the donor nucleic acidare located together in a rationally-designed editing cassette where atleast two editing cassettes in an editing vector or plasmid aresimultaneously transformed into a bacteria cell.

The target sequence is associated with a proto-spacer adjacent motif(PAM), which is a short nucleotide sequence recognized by thegRNA/nuclease complex. The precise preferred PAM sequence and lengthrequirements for different nucleic acid-guided nucleases vary; however,PAMs typically are 2-7 base-pair sequences adjacent or in proximity tothe target sequence and, depending on the nuclease, can be 5′ or 3′ tothe target sequence. Engineering of the PAM-interacting domain of anucleic acid-guided nuclease may allow for alteration of PAMspecificity, improve target site recognition fidelity, decrease targetsite recognition fidelity, or increase the versatility of a nucleicacid-guided nuclease.

In most embodiments, genome editing of a cellular target sequence bothintroduces a desired DNA change to a cellular target sequence, e.g., thegenomic DNA of a cell, and removes, mutates, or renders inactive aproto-spacer adjacent motif (PAM) and/or spacer region in the cellulartarget sequence (e.g., renders the target site immune to furthernuclease binding). Rendering the PAM at the cellular target sequenceinactive precludes additional editing of the cell genome at thatcellular target sequence, e.g., upon subsequent exposure to a nucleicacid-guided nuclease complexed with a synthetic guide nucleic acid inlater rounds of editing. Thus, cells having the desired cellular targetsequence edit and an altered PAM can be selected for by using a nucleicacid-guided nuclease complexed with a synthetic guide nucleic acidcomplementary to the cellular target sequence. Cells that did notundergo the first editing event will be cut rendering a double-strandedDNA break, and thus will not continue to be viable. The cells containingthe desired cellular target sequence edit and PAM alteration will not becut, as these edited cells no longer contain the necessary PAM site andwill continue to grow and propagate.

As for the nuclease component of the nucleic acid-guided nucleaseediting system, a polynucleotide sequence encoding the nucleicacid-guided nuclease can be codon optimized for expression in particularcell types, such as bacteria cells. The choice of nucleic acid-guidednuclease to be employed depends on many factors, such as what type ofedit is to be made in the target sequence and whether an appropriate PAMis located close to the desired target sequence. Nucleases of use in themethods described herein include but are not limited to Cas 9, Cas12/CpfI, MAD2, or MAD7 or other MADzymes. As with the guide nucleicacid, the nuclease is encoded by a DNA sequence on a vector andoptionally is under the control of an inducible promoter. In someembodiments, the promoter may be separate from but the same as thepromoter controlling transcription of the guide nucleic acid; that is, aseparate promoter drives the transcription of the nuclease and guidenucleic acid sequences but the two promoters may be the same type ofpromoter. Alternatively, the promoter controlling expression of thenuclease may be different from the promoter controlling transcription ofthe guide nucleic acid; that is, e.g., the nuclease may be under thecontrol of, e.g., the p1 promoter, and the guide nucleic acid may beunder the control of the, e.g., pBAD promoter.

Another component of the nucleic acid-guided nuclease system is thedonor nucleic acid (or homology arm) comprising homology to the cellulartarget sequence. The donor nucleic acid is on the same vector and evenin the same editing cassette as the guide nucleic acid and preferably is(but not necessarily is) under the control of the same promoter as theediting gRNA (that is, a single promoter driving the transcription ofboth the editing gRNA and the donor nucleic acid). The donor nucleicacid is designed to serve as a template for homologous recombinationwith a cellular target sequence nicked or cleaved by the nucleicacid-guided nuclease as a part of the gRNA/nuclease complex; that is,the donor nucleic acid provides the desired edit in the cellular targetsequence. A donor nucleic acid polynucleotide may be of any suitablelength, such as about or more than about 20, 25, 50, 75, 100, 150, 200,500, or 1000 nucleotides in length, and up to 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13 and up to 20 kb in length if combined with a dual gRNAarchitecture as described in U.S. Ser. No. 16/275,465, filed 14 Feb.2019. In certain preferred aspects, the donor nucleic acid can beprovided as an oligonucleotide of between 20-300 nucleotides, morepreferably between 50-250 nucleotides. The donor nucleic acid comprisesa region that is complementary to a portion of the cellular targetsequence (e.g., a homology arm). When optimally aligned, the donornucleic acid overlaps with (is complementary to) the cellular targetsequence by, e.g., about 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or morenucleotides. In many embodiments, the donor nucleic acid comprises twohomology arms (regions complementary to the cellular target sequence)flanking the mutation or difference between the donor nucleic acid andthe cellular target sequence. The donor nucleic acid comprises at leastone mutation or alteration compared to the cellular target sequence,such as an insertion, deletion, modification, or any combination thereofcompared to the cellular target sequence.

As described in relation to the gRNA, the donor nucleic acid ispreferably provided as part of a rationally-designed editing cassette,where, in this embodiment, two of which are inserted into an editingplasmid backbone where the editing plasmid backbone may comprise apromoter to drive transcription of the editing cassettes. In the methodembodiments described herein there may be more than two, e.g., three,four, or more editing gRNA/donor nucleic acid rationally-designedediting cassettes inserted into an editing vector, where each editingcassette is under the control of separate, different promoters, or eachediting cassette is under the control of separate like promoters, orwhere all editing cassettes are under the control of a single promoter.In some embodiments the promoter driving transcription of the editingcassettes is optionally an inducible promoter.

In addition to the donor nucleic acid, an editing cassette may compriseone or more primer sites. The primer sites can be used to amplify theediting cassettes by using oligonucleotide primers; for example, if theprimer sites flank one or more of the other components of the editingcassette. In addition, the editing cassettes may comprise a barcode. Abarcode is a unique DNA sequence that corresponds to the donor DNAsequence such that the barcode can identify the edit made to thecorresponding cellular target sequence. The barcode typically comprisesfour or more nucleotides and is captured to determine what edit was madein the single cell workflow. In some embodiments, the editing cassettescomprise a collection or library of editing gRNAs and of donor nucleicacids representing, e.g., gene-wide or genome-wide libraries of editinggRNAs and donor nucleic acids. The library of editing cassettes iscloned into vector backbones where, e.g., each different donor nucleicacid is associated with a different barcode. Also, in preferredembodiments, an editing vector or plasmid encoding components of thenucleic acid-guided nuclease system further encodes a nucleicacid-guided nuclease comprising one or more nuclear localizationsequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or more NLSs, particularly as an element of the nucleasesequence. In some embodiments, the engineered nuclease comprises NLSs ator near the amino-terminus, NLSs at or near the carboxy-terminus, or acombination.

Increasing Observed Editing Rates of Transformed Bacteria Cells

The present disclosure is drawn to increasing the observed editing ratesin bacteria cells post-editing. The present compositions and methods incombination lead to a phenomenon of “edit or die.” Although less cellssurvive plating and editing, a large percentage of cells that do surviveare multiple editors. In one experiment it was found that if a cellsurvives transformation, plating and editing, 75% of the surviving cellsare triple editors; that is, 75% of the surviving cells weresimultaneously edited with edits at three different locations within thebacterial genome.

FIG. 1A is a general flow chart for the nucleic guided-nuclease editingmethods according to the present disclosure. In a first step of method100, a library of rationally-designed editing cassettes is synthesized102, where at least two editing cassettes targeting two different targetregions in the bacterial genome are used in each editing plasmid orvector. Again, methods and compositions for designing and synthesizingediting cassettes are described in U.S. Pat. Nos. 10,240,167;10,266,849; 9,982,278; 10,351,877; 10,364,442; 10,435,715; and10,465,207, all of which are incorporated herein in their entirety. U.S.Pat. No. 10,465,207 is drawn to multiplex or compound editing cassettes(e.g., two or more cassettes targeting different regions in the genome)as used in the method embodiments employed herein. Once designed andsynthesized, the editing cassettes are amplified and purified. Oneexemplary multiplex or compound editing cassette is shown in FIG. 1E.

Next or simultaneously at step 104, editing plasmid backbones aredesigned. The editing plasmid backbone typically comprises at least oneselectable marker sequence, a bacterial origin of replication and othergenetic elements. Also next or simultaneously, an engine plasmid isdesigned and synthesized at step 118. Exemplary engine plasmids areshown at FIGS. 1B, 1C and 1D and comprise a coding sequence for anucleic acid-guided nuclease (e.g., a coding sequence for MAD7), thecoding sequences for the components of the Red recombineering system, abacterial origin of replication, a selectable marker sequence (e.g.,typically a different selectable marker than the selectable marker thatis on the editing plasmid), and, if present, a coding sequence for therecA protein or recA fusion protein.

In addition to preparing editing and engine plasmids, the bacterialcells of choice are made electrocompetent 120 for transformation. Oncethe bacterial cells are rendered electrocompetent 120, the cells,editing plasmids, and engine plasmids are combined and the editingplasmids and engine plasmids are transformed into (e.g., electroporatedinto) the cells 106. In some embodiments of the present methods, thecells are transformed simultaneously with an editing plasmid and anengine plasmid expressing the editing nuclease; alternatively, the cellsmay already have been transformed with an engine plasmid configured toexpress the nuclease. Transformation is intended to include to a varietyof art-recognized techniques for introducing an exogenous nucleic acidsequence (e.g., DNA) into a target cell, and the term “transformation”as used herein includes all transformation and transfection techniques.Such methods include, but are not limited to, electroporation,lipofection, optoporation, injection, microprecipitation,microinjection, liposomes, particle bombardment, sonoporation,laser-induced poration, bead transfection, calcium phosphate or calciumchloride co-precipitation, or DEAE-dextran-mediated transfection.Additionally, hybrid techniques that exploit the capabilities ofmechanical and chemical transfection methods can be used, e.g.,magnetofection, a transfection methodology that combines chemicaltransfection with mechanical methods. In another example, cationiclipids may be deployed in combination with gene guns or electroporators.Suitable materials and methods for transforming or transfecting targetcells can be found, e.g., in Green and Sambrook, Molecular Cloning: ALaboratory Manual, 4th, ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 2014). The present automated methods using theautomated multi-module cell processing instrument utilize flow-throughelectroporation such as the exemplary device shown in FIGS. 7A-7E.

Once transformed, the cells are allowed to edit 108 in the presence ofselective agents that select for the engine and editing plasmids. Asdescribed above, drug selectable markers such asampilcillin/carbenicillin, kanamycin, chloramphenicol, nourseothricinN-acetyl transferase, erythromycin, tetracycline, gentamicin, bleomycin,streptomycin, puromycin or other selectable markers may be employed.

At a next step, after editing takes place the cells are grown 112until 1) the cells enter (or are close to entering) the stationary phaseof growth and colonies become normalized or 2) until the cells formdifferentially-sized colonies (e.g., large and small colonies). If cellsare grown until the colonies are normalized (e.g., all cells enter thestationary phase of growth), the cells will be enriched for editedcells. If the cells are grown to where differently-sized colonies arepresented, small colonies can be selected where the small colonies arelikely colonies arising from edited cells. See, e.g., U.S. Ser. No.16/454,865, filed 26 Jun. 2019 and U.S. Ser. No. 16/597,831, filed 9Oct. 2019. Once the cells are grown either to stationary phase or todifferentially-sized colonies, the cells may be pooled (if fromnormalized colonies) or selected (if from differentially-sized colonies)and transferred to a different vessel and fresh media, then grown to adesired OD to be made electrocompetent 114 again, followed by anotherround of editing 116.

FIG. 1B is an exemplary engine vector map for the p197 engine vector.Beginning at approximately 10 o'clock, there is a pBAD induciblepromoter driving transcription of the Red recombineering system; anSC101 bacterial origin of replication (in this instance, a temperaturesensitive origin of replication); a coding sequence for the c1857repressor gene (described below); a coding sequence for a carbenicillinresistance gene; and a pL inducible promoter driving expression of aMAD7 nuclease coding sequence. The protein product of the c1857repressor gene on the engine vector at temperatures under 40° C.actively represses the pL promoter driving transcription of thenuclease; however, at temperatures above 40° C. the protein product ofthe c1857 repressor gene on the engine vector unfolds (e.g., degrades).The unfolded or degraded c1857 repressor gene protein product cannotbind the pL promoter driving expression of the nuclease and thus the pLpromoter is active driving transcription of the nuclease on the enginevector at elevated temperatures.

FIG. 1C is an exemplary engine vector map for the p197-recA enginevector. Beginning at approximately 10 o'clock, there is a pBAD induciblepromoter driving transcription of the Red recombineering system; anSC101 bacterial origin of replication (in this instance, a temperaturesensitive origin of replication); a coding sequence for a recA protein(described below); a coding sequence for the c1857 repressor gene(described above); a coding sequence for a carbenicillin resistancegene; and a pL inducible promoter driving expression of a MAD7 nucleasecoding sequence. As with the p197 engine vector described in relation toFIG. 1B, the protein product of the c1857 repressor gene on the enginevector at temperatures under 40° C. actively represses the pL promoterhalting transcription of the nuclease but at temperatures above 42° C.the protein product of the c1857 repressor gene degrades and is unableto repress the pL promoter thereby allowing expression of the MAD7nuclease. As for recA, recA is a 38 kilodalton protein that is essentialfor the repair and maintenance of DNA. RecA has been found to have astructural and functional homolog in every species examined. Thehomologous protein in eukaryotes is RAD51. In the embodiments herein,recA is used to increase DNA repair as a result of the editing process.

FIG. 1D is an exemplary engine vector map for the p197-srpRrecA enginevector. Beginning at approximately 10 o'clock, there is a pBAD induciblepromoter driving transcription of the λRed recombineering system; anSC101 bacterial origin of replication (in this instance, a temperaturesensitive origin of replication); a coding sequence for an srpP proteincoding sequence (described below) fused to a recA protein (describedabove); a coding sequence for the c1857 repressor gene (describedabove); a coding sequence for a carbenicillin resistance gene and a pLinducible promoter driving expression of a MAD7 nuclease codingsequence. As with the p197 engine vector described in relation to FIG.1B and p197-recA vector described in relation to FIG. 1C, the proteinproduct of the c1857 repressor gene on the engine vector at temperaturesunder 40° C. actively represses the pL promoter halting transcription ofthe nuclease but at temperatures above 42° C. the protein product of thec1857 repressor gene degrades thereby allowing expression of the MAD7nuclease. As for recA, as described above, recA is a protein essentialfor the repair and maintenance of DNA. In the p197-recAsrpR enginevector, the recA coding sequence is fused to an srpR coding sequence viaan 18-bp linker sequence. The stop codon (TAA) of the srpR codingsequence was removed to create an in-frame fusion protein comprising thelast amino acid (glutamate) of the srpR protein at the N-terminalportion and the first amino acid (methionine) of the recA protein. ThesrpR protein is a DNA binding protein originally isolated fromPseudomonas putida, and putatively encodes a TetR family repressor withstrong DNA binding affinity (see, e.g., Stanton, et al., “Genomic miningof prokaryotic repressors for orthogonal logic gates”, Nat. Chem. Biol.,10:99-105 (2014)).

In addition to one of the engine plasmids described in relation to FIGS.1B-1D, an editing plasmid is also transformed into the bacteria cells ofinterest. As described above, the editing plasmid comprises at least twogRNA/donor DNA (homology arm) pairs targeting two different targetregions in the bacterial genome; and in one exemplary editing plasmid asdepicted in FIG. 1E, the editing plasmid comprises three gRNA/donor DNA(homology arm) pairs targeting three different target regions in thebacterial genome. Again, methods and compositions for designing andsynthesizing editing cassettes are described in U.S. Pat. Nos.10,240,167; 10,266,849; 9,982,278; 10,351,877; 10,364,442; 10,435,715;and 10,465,207, all of which are incorporated herein in their entirety;where U.S. Pat. No. 10,465,207 is drawn to multiplex or compound editingcassettes (e.g., two or more cassettes targeting different regions inthe genome) as used in the method embodiments employed herein. Lookingat FIG. 1E, beginning at approximately 10 o'clock, there is a promoterdriving expression of a selection marker (e.g., antibiotic resistancegene, and preferably not the same antibiotic resistance gene as islocated on the engine plasmid); a pL inducible promoter drivingexpression of the three editing cassettes (e.g., CR-SR1-HA1-L1;CR-SR2-HA2-L2; and CR-SR3-HA3-L3) (the p1 promoter and control thereofby the c1857 repressor gene on the engine vector is described above);and a bacterial origin of replication. The components of the editingcassettes include “CR”, which is the portion of the gRNA correspondingto the CRISPR structure sequence for the MAD7 nuclease, where eachediting cassette comprises the same or very similar CR sequence; “SR”,which is the target-specific spacer region of the gRNA and is differentfor each editing cassette in this example; “HA”, which is the homologyarm or donor DNA component and is different for each editing cassette inthis example; and “L”, which is a linker sequence that is different foreach editing cassette and in this example is used in assembly of themultiplex (e.g., compound) editing cassette.

Automated Cell Editing Instruments and Modules to Perform NucleicAcid-Guided Nuclease Editing in Cells

Automated Cell Editing Instruments

FIG. 2A depicts an exemplary automated multi-module cell processinginstrument 200 to, e.g., perform rationally-designed genome edits inbacteria as described herein. The instrument 200, for example, may beand preferably is designed as a stand-alone desktop instrument for usewithin a laboratory environment. The instrument 200 may incorporate amixture of reusable and disposable components for performing the variousintegrated processes in conducting automated genome cleavage and/orediting in cells without human intervention. Illustrated is a gantry202, providing an automated mechanical motion system (actuator) (notshown) that supplies XYZ axis motion control to, e.g., an automated(i.e., robotic) liquid handling system 258 including, e.g., an airdisplacement pipettor 232 which allows for cell processing amongmultiple modules without human intervention. In some automatedmulti-module cell processing instruments, the air displacement pipettor232 is moved by gantry 202 and the various modules and reagentcartridges remain stationary; however, in other embodiments, the liquidhandling system 258 may stay stationary while the various modules andreagent cartridges are moved. Also included in the automatedmulti-module cell processing instrument 200 are reagent cartridges 210comprising reservoirs 212 and transformation module 230 (e.g., aflow-through electroporation device as described in detail in relationto FIGS. 5B-5F), as well as wash reservoirs 206, cell input reservoir251 and cell output reservoir 253. The wash reservoirs 206 may beconfigured to accommodate large tubes, for example, wash solutions, orsolutions that are used often throughout an iterative process. Althoughtwo of the reagent cartridges 210 comprise a wash reservoir 206 in FIG.2A, the wash reservoirs instead could be included in a wash cartridgewhere the reagent and wash cartridges are separate cartridges. In such acase, the reagent cartridge 210 and wash cartridge 204 may be identicalexcept for the consumables (reagents or other components containedwithin the various inserts) inserted therein.

In some implementations, the reagent cartridges 210 are disposable kitscomprising reagents and cells for use in the automated multi-module cellprocessing/editing instrument 200. For example, a user may open andposition each of the reagent cartridges 210 comprising various desiredinserts and reagents within the chassis of the automated multi-modulecell editing instrument 200 prior to activating cell processing.Further, each of the reagent cartridges 210 may be inserted intoreceptacles in the chassis having different temperature zonesappropriate for the reagents contained therein.

Also illustrated in FIG. 2A is the robotic liquid handling system 258including the gantry 202 and air displacement pipettor 232. In someexamples, the robotic handling system 258 may include an automatedliquid handling system such as those manufactured by Tecan Group Ltd. ofMannedorf, Switzerland, Hamilton Company of Reno, Nev. (see, e.g.,WO2018015544A1), or Beckman Coulter, Inc. of Fort Collins, Colo. (see,e.g., US20160018427A1). Pipette tips may be provided in a pipettetransfer tip supply (not shown) for use with the air displacementpipettor 232.

Inserts or components of the reagent cartridges 210, in someimplementations, are marked with machine-readable indicia (not shown),such as bar codes, for recognition by the robotic handling system 258.For example, the robotic liquid handling system 258 may scan one or moreinserts within each of the reagent cartridges 210 to confirm contents.In other implementations, machine-readable indicia may be marked uponeach reagent cartridge 210, and a processing system (not shown, but seeelement 237 of FIG. 2B) of the automated multi-module cell editinginstrument 200 may identify a stored materials map based upon themachine-readable indicia. In the embodiment illustrated in FIG. 2A, acell growth module comprises two cell growth vials 218, 220 (describedin greater detail below in relation to FIGS. 3A-3D). Additionally seenis the TFF module 222 (described above in detail in relation to FIGS.4A-4E). Also illustrated as part of the automated multi-module cellprocessing instrument 200 of FIG. 2A is a singulation module 240 (e.g.,a solid wall isolation, incubation and normalization device (SWIINdevice) is shown here) described herein in relation to FIGS. 6B-6E,served by, e.g., robotic liquid handing system 258 and air displacementpipettor 232. Also note the placement of three heatsinks 255.

FIG. 2B is a simplified representation of the contents of the exemplarymulti-module cell processing instrument 200 depicted in FIG. 2A.Cartridge-based source materials (such as in reagent cartridges 210),for example, may be positioned in designated areas on a deck of theinstrument 200 for access by an air displacement pipettor 232. The deckof the multi-module cell processing instrument 200 may include aprotection sink such that contaminants spilling, dripping, oroverflowing from any of the modules of the instrument 200 are containedwithin a lip of the protection sink. Also seen are reagent cartridges210, which are shown disposed with thermal assemblies 211 which cancreate temperature zones appropriate for different regions. Note thatone of the reagent cartridges also comprises a flow-throughelectroporation device 230 (FTEP), served by FTEP interface (e.g.,manifold arm) and actuator 231. Also seen is TFF module 222 withadjacent thermal assembly 225, where the TFF module is served by TFFinterface (e.g., manifold arm) and actuator 233. Thermal assemblies 225,235, and 245 encompass thermal electric devices such as Peltier devices,as well as heatsinks, fans and coolers. The two rotating growth vials218 and 220 are within a growth module 234, where the growth module isserved by two thermal assemblies 235. Also seen is the SWIIN module 240,comprising a SWIIN cartridge 241, where the SWIIN module also comprisesa thermal assembly 245, illumination 243 (in this embodiment,backlighting), evaporation and condensation control 249, and where theSWIIN module is served by SWIIN interface (e.g., manifold arm) andactuator 247.

Also seen in this view is touch screen display 201, display actuator203, illumination 205 (one on either side of multi-module cellprocessing instrument 200), and cameras 239 (one illumination device oneither side of multi-module cell processing instrument 200). Finally,element 237 comprises electronics, such as circuit control boards,high-voltage amplifiers, power supplies, and power entry; as well aspneumatics, such as pumps, valves and sensors.

FIG. 2C illustrates a front perspective (door open) view of multi-modulecell processing instrument 200 in a desktop version of the automatedmulti-module cell editing instrument 200. For example, a chassis 290 mayhave a width of about 24-48 inches, a height of about 24-48 inches and adepth of about 24-48 inches. Chassis 290 may be and preferably isdesigned to hold all modules and disposable supplies used in automatedcell processing and to perform all processes required without humanintervention; that is, chassis 290 is configured to provide anintegrated, stand-alone automated multi-module cell processinginstrument. As illustrated in FIG. 2C, chassis 290 includes touch screendisplay 201, cooling grate 264, which allows for air flow via aninternal fan (not shown). The touch screen display provides informationto a user regarding the processing status of the automated multi-modulecell editing instrument 200 and accepts inputs from the user forconducting the cell processing. In this embodiment, the chassis 290 islifted by adjustable feet 270 a, 270 b, 270 c and 270 d (feet 270 a-270c are shown in this FIG. 2C). Adjustable feet 270 a-270 d, for example,allow for additional air flow beneath the chassis 290.

Inside the chassis 290, in some implementations, will be most or all ofthe components described in relation to FIGS. 2A and 2B, including therobotic liquid handling system disposed along a gantry, reagentcartridges 210 including a flow-through electroporation device, one ormore rotating growth vials 218, 220 in a cell growth module 234, atangential flow filtration module 222, a SWIIN module 240 as well asinterfaces and actuators for the various modules. In addition, chassis290 houses control circuitry, liquid handling tubes, air pump controls,valves, sensors, thermal assemblies (e.g., heating and cooling units)and other control mechanisms. For examples of multi-module cell editinginstruments, see U.S. Pat. No. 10,253,316, issued 9 Apr. 2019; U.S. Pat.No. 10,329,559, issued 25 Jun. 2019; and U.S. Pat. No. 10,323,242,issued 18 Jun. 2019; U.S. Pat. No. 10,421,959, issued 24 Sep. 2019; andSer. No. 16/412,195, filed 14 May 2019; and Ser. No. 16/423,289, filed28 May 2019, all of which are herein incorporated by reference in theirentirety.

The Rotating Cell Growth Module

FIG. 3A shows one embodiment of a rotating growth vial 300 for use withthe cell growth device and in the automated multi-module cell processinginstruments described herein. The rotating growth vial 300 is anoptically-transparent container having an open end 304 for receivingliquid media and cells, a central vial region 306 that defines theprimary container for growing cells, a tapered-to-constricted region 318defining at least one light path 310, a closed end 316, and a driveengagement mechanism 312. The rotating growth vial 300 has a centrallongitudinal axis 320 around which the vial rotates, and the light path310 is generally perpendicular to the longitudinal axis of the vial. Thefirst light path 310 is positioned in the lower constricted portion ofthe tapered-to-constricted region 318. Optionally, some embodiments ofthe rotating growth vial 300 have a second light path 308 in the taperedregion of the tapered-to-constricted region 318. Both light paths inthis embodiment are positioned in a region of the rotating growth vialthat is constantly filled with the cell culture (cells+growth media) andare not affected by the rotational speed of the growth vial. The firstlight path 310 is shorter than the second light path 308 allowing forsensitive measurement of OD values when the OD values of the cellculture in the vial are at a high level (e.g., later in the cell growthprocess), whereas the second light path 308 allows for sensitivemeasurement of OD values when the OD values of the cell culture in thevial are at a lower level (e.g., earlier in the cell growth process).

The drive engagement mechanism 312 engages with a motor (not shown) torotate the vial. In some embodiments, the motor drives the driveengagement mechanism 312 such that the rotating growth vial 300 isrotated in one direction only, and in other embodiments, the rotatinggrowth vial 300 is rotated in a first direction for a first amount oftime or periodicity, rotated in a second direction (i.e., the oppositedirection) for a second amount of time or periodicity, and this processmay be repeated so that the rotating growth vial 300 (and the cellculture contents) are subjected to an oscillating motion. Further, thechoice of whether the culture is subjected to oscillation and theperiodicity therefor may be selected by the user. The first amount oftime and the second amount of time may be the same or may be different.The amount of time may be 1, 2, 3, 4, 5, or more seconds, or may be 1,2, 3, 4 or more minutes. In another embodiment, in an early stage ofcell growth the rotating growth vial 400 may be oscillated at a firstperiodicity (e.g., every 60 seconds), and then a later stage of cellgrowth the rotating growth vial 300 may be oscillated at a secondperiodicity (e.g., every one second) different from the firstperiodicity.

The rotating growth vial 300 may be reusable or, preferably, therotating growth vial is consumable. In some embodiments, the rotatinggrowth vial is consumable and is presented to the user pre-filled withgrowth medium, where the vial is hermetically sealed at the open end 304with a foil seal. A medium-filled rotating growth vial packaged in sucha manner may be part of a kit for use with a stand-alone cell growthdevice or with a cell growth module that is part of an automatedmulti-module cell processing system. To introduce cells into the vial, auser need only pipette up a desired volume of cells and use the pipettetip to punch through the foil seal of the vial. Open end 304 mayoptionally include an extended lip 402 to overlap and engage with thecell growth device. In automated systems, the rotating growth vial 400may be tagged with a barcode or other identifying means that can be readby a scanner or camera (not shown) that is part of the automated system.

The volume of the rotating growth vial 300 and the volume of the cellculture (including growth medium) may vary greatly, but the volume ofthe rotating growth vial 300 must be large enough to generate aspecified total number of cells. In practice, the volume of the rotatinggrowth vial 400 may range from 1-250 mL, 2-100 mL, from 5-80 mL, 10-50mL, or from 12-35 mL. Likewise, the volume of the cell culture(cells+growth media) should be appropriate to allow proper aeration andmixing in the rotating growth vial 400. Proper aeration promotes uniformcellular respiration within the growth media. Thus, the volume of thecell culture should be approximately 5-85% of the volume of the growthvial or from 20-60% of the volume of the growth vial. For example, for a30 mL growth vial, the volume of the cell culture would be from about1.5 mL to about 26 mL, or from 6 mL to about 18 mL.

The rotating growth vial 300 preferably is fabricated from abio-compatible optically transparent material—or at least the portion ofthe vial comprising the light path(s) is transparent. Additionally,material from which the rotating growth vial is fabricated should beable to be cooled to about 4° C. or lower and heated to about 55° C. orhigher to accommodate both temperature-based cell assays and long-termstorage at low temperatures. Further, the material that is used tofabricate the vial must be able to withstand temperatures up to 55° C.without deformation while spinning. Suitable materials include cyclicolefin copolymer (COC), glass, polyvinyl chloride, polyethylene,polyamide, polypropylene, polycarbonate, poly(methyl methacrylate(PMMA), polysulfone, polyurethane, and co-polymers of these and otherpolymers. Preferred materials include polypropylene, polycarbonate, orpolystyrene. In some embodiments, the rotating growth vial isinexpensively fabricated by, e.g., injection molding or extrusion.

FIG. 3B is a perspective view of one embodiment of a cell growth device330. FIG. 3C depicts a cut-away view of the cell growth device 330 fromFIG. 3B. In both figures, the rotating growth vial 300 is seenpositioned inside a main housing 336 with the extended lip 302 of therotating growth vial 300 extending above the main housing 336.Additionally, end housings 352, a lower housing 332 and flanges 334 areindicated in both figures. Flanges 334 are used to attach the cellgrowth device 330 to heating/cooling means or other structure (notshown). FIG. 3C depicts additional detail. In FIG. 3C, upper bearing 342and lower bearing 340 are shown positioned within main housing 336.Upper bearing 342 and lower bearing 340 support the vertical load ofrotating growth vial 300. Lower housing 332 contains the drive motor338. The cell growth device 330 of FIG. 3C comprises two light paths: aprimary light path 344, and a secondary light path 350. Light path 344corresponds to light path 310 positioned in the constricted portion ofthe tapered-to-constricted portion of the rotating growth vial 300, andlight path 350 corresponds to light path 308 in the tapered portion ofthe tapered-to-constricted portion of the rotating growth via 316. Lightpaths 310 and 308 are not shown in FIG. 3C but may be seen in FIG. 3A.In addition to light paths 344 and 340, there is an emission board 348to illuminate the light path(s), and detector board 346 to detect thelight after the light travels through the cell culture liquid in therotating growth vial 300.

The motor 338 engages with drive mechanism 312 and is used to rotate therotating growth vial 300. In some embodiments, motor 338 is a brushlessDC type drive motor with built-in drive controls that can be set to holda constant revolution per minute (RPM) between 0 and about 3000 RPM.Alternatively, other motor types such as a stepper, servo, brushed DC,and the like can be used. Optionally, the motor 338 may also havedirection control to allow reversing of the rotational direction, and atachometer to sense and report actual RPM. The motor is controlled by aprocessor (not shown) according to, e.g., standard protocols programmedinto the processor and/or user input, and the motor may be configured tovary RPM to cause axial precession of the cell culture thereby enhancingmixing, e.g., to prevent cell aggregation, increase aeration, andoptimize cellular respiration.

Main housing 336, end housings 352 and lower housing 332 of the cellgrowth device 330 may be fabricated from any suitable, robust materialincluding aluminum, stainless steel, and other thermally conductivematerials, including plastics. These structures or portions thereof canbe created through various techniques, e.g., metal fabrication,injection molding, creation of structural layers that are fused, etc.Whereas the rotating growth vial 300 is envisioned in some embodimentsto be reusable, but preferably is consumable, the other components ofthe cell growth device 330 are preferably reusable and function as astand-alone benchtop device or as a module in a multi-module cellprocessing system.

The processor (not shown) of the cell growth device 330 may beprogrammed with information to be used as a “blank” or control for thegrowing cell culture. A “blank” or control is a vessel containing cellgrowth medium only, which yields 100% transmittance and 0 OD, while thecell sample will deflect light rays and will have a lower percenttransmittance and higher OD. As the cells grow in the media and becomedenser, transmittance will decrease and OD will increase. The processor(not shown) of the cell growth device 330—may be programmed to usewavelength values for blanks commensurate with the growth mediatypically used in cell culture (whether, e.g., mammalian cells,bacterial cells, animal cells, yeast cells, etc.). Alternatively, asecond spectrophotometer and vessel may be included in the cell growthdevice 330, where the second spectrophotometer is used to read a blankat designated intervals.

FIG. 3D illustrates a cell growth device 330 as part of an assemblycomprising the cell growth device 330 of FIG. 3B coupled to light source390, detector 392, and thermal components 394. The rotating growth vial300 is inserted into the cell growth device. Components of the lightsource 390 and detector 392 (e.g., such as a photodiode with gaincontrol to cover 5-log) are coupled to the main housing of the cellgrowth device. The lower housing 332 that houses the motor that rotatesthe rotating growth vial 300 is illustrated, as is one of the flanges334 that secures the cell growth device 330 to the assembly. Also, thethermal components 394 illustrated are a Peltier device orthermoelectric cooler. In this embodiment, thermal control isaccomplished by attachment and electrical integration of the cell growthdevice 330 to the thermal components 394 via the flange 334 on the baseof the lower housing 332. Thermoelectric coolers are capable of“pumping” heat to either side of a junction, either cooling a surface orheating a surface depending on the direction of current flow. In oneembodiment, a thermistor is used to measure the temperature of the mainhousing and then, through a standard electronicproportional-integral-derivative (PID) controller loop, the rotatinggrowth vial 300 is controlled to approximately +/−0.5° C.

In use, cells are inoculated (cells can be pipetted, e.g., from anautomated liquid handling system or by a user) into pre-filled growthmedia of a rotating growth vial 300 by piercing though the foil seal orfilm. The programmed software of the cell growth device 330 sets thecontrol temperature for growth, typically 30° C., then slowly starts therotation of the rotating growth vial 300. The cell/growth media mixtureslowly moves vertically up the wall due to centrifugal force allowingthe rotating growth vial 300 to expose a large surface area of themixture to a normal oxygen environment. The growth monitoring systemtakes either continuous readings of the OD or OD measurements at pre-setor pre-programmed time intervals. These measurements are stored ininternal memory and if requested the software plots the measurementsversus time to display a growth curve. If enhanced mixing is required,e.g., to optimize growth conditions, the speed of the vial rotation canbe varied to cause an axial precession of the liquid, and/or a completedirectional change can be performed at programmed intervals. The growthmonitoring can be programmed to automatically terminate the growth stageat a pre-determined OD, and then quickly cool the mixture to a lowertemperature to inhibit further growth.

One application for the cell growth device 330 is to constantly measurethe optical density of a growing cell culture. One advantage of thedescribed cell growth device is that optical density can be measuredcontinuously (kinetic monitoring) or at specific time intervals; e.g.,every 5, 10, 15, 20, 30 45, or 60 seconds, or every 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 minutes. While the cell growth device 330 has been describedin the context of measuring the optical density (OD) of a growing cellculture, it should, however, be understood by a skilled artisan giventhe teachings of the present specification that other cell growthparameters can be measured in addition to or instead of cell culture OD.As with optional measure of cell growth in relation to the solid walldevice or module described supra, spectroscopy using visible, UV, ornear infrared (NIR) light allows monitoring the concentration ofnutrients and/or wastes in the cell culture and other spectroscopicmeasurements may be made; that is, other spectral properties can bemeasured via, e.g., dielectric impedance spectroscopy, visiblefluorescence, fluorescence polarization, or luminescence. Additionally,the cell growth device 430 may include additional sensors for measuring,e.g., dissolved oxygen, carbon dioxide, pH, conductivity, and the like.For additional details regarding rotating growth vials and cell growthdevices see U.S. Pat. No. 10,435,662, issued 8 Oct. 2019; and U.S. Pat.No. 10,443,031, issued 15 Oct. 2019; and U.S. Ser. No. 16/552,981, filed27 Aug. 2019.

The Cell Concentration Module

As described above in relation to the rotating growth vial and cellgrowth module, in order to obtain an adequate number of cells fortransformation or transfection, cells typically are grown to a specificoptical density in medium appropriate for the growth of the cells ofinterest; however, for effective transformation or transfection, it isdesirable to decrease the volume of the cells as well as render thecells competent via buffer or medium exchange. Thus, one sub-componentor module that is desired in cell processing systems for the processeslisted above is a module or component that can grow, perform bufferexchange, and/or concentrate cells and render them competent so thatthey may be transformed or transfected with the nucleic acids needed forengineering or editing the cell's genome.

FIG. 4A shows a retentate member 422 (top), permeate member 420 (middle)and a tangential flow assembly 410 (bottom) comprising the retentatemember 422, membrane 424 (not seen in FIG. 4A), and permeate member 420(also not seen). In FIG. 4A, retentate member 422 comprises a tangentialflow channel 402, which has a serpentine configuration that initiates atone lower corner of retentate member 422—specifically at retentate port428—traverses across and up then down and across retentate member 422,ending in the other lower corner of retentate member 422 at a secondretentate port 428. Also seen on retentate member 422 are energydirectors 491, which circumscribe the region where a membrane or filter(not seen in this FIG. 4A) is seated, as well as interdigitate betweenareas of channel 402. Energy directors 491 in this embodiment mate withand serve to facilitate ultrasonic welding or bonding of retentatemember 422 with permeate/filtrate member 420 via the energy directorcomponent 491 on permeate/filtrate member 420 (at right). Additionally,countersinks 423 can be seen, two on the bottom one at the top center ofretentate member 422. Countersinks 423 are used to couple and tangentialflow assembly 410 to a reservoir assembly (not seen in this FIG. 4A butsee FIG. 4B).

Permeate/filtrate member 420 is seen in the middle of FIG. 4A andcomprises, in addition to energy director 491, through-holes forretentate ports 428 at each bottom corner (which mate with thethrough-holes for retentate ports 428 at the bottom corners of retentatemember 422), as well as a tangential flow channel 402 and twopermeate/filtrate ports 426 positioned at the top and center of permeatemember 420. The tangential flow channel 402 structure in this embodimenthas a serpentine configuration and an undulating geometry, althoughother geometries may be used. Permeate member 420 also comprisescountersinks 423, coincident with the countersinks 423 on retentatemember 420.

At bottom of FIG. 4A is a tangential flow assembly 410 comprising theretentate member 422 and permeate member 420 seen in this FIG. 4A. Inthis view, retentate member 422 is “on top” of the view, a membrane (notseen in this view of the assembly) would be adjacent and under retentatemember 422 and permeate member 420 (also not seen in this view of theassembly) is adjacent to and beneath the membrane. Again countersinks423 are seen, where the countersinks in the retentate member 422 and thepermeate member 420 are coincident and configured to mate with threadsor mating elements for the countersinks disposed on a reservoir assembly(not seen in FIG. 4A but see FIG. 4B).

A membrane or filter is disposed between the retentate and permeatemembers, where fluids can flow through the membrane but cells cannot andare thus retained in the flow channel disposed in the retentate member.Filters or membranes appropriate for use in the TFF device/module arethose that are solvent resistant, are contamination free duringfiltration, and are able to retain the types and sizes of cells ofinterest. For example, in order to retain small cell types such asbacterial cells, pore sizes can be as low as 0.2 μm, however for othercell types, the pore sizes can be as high as 20 μm. Indeed, the poresizes useful in the TFF device/module include filters with sizes from0.20 μm, 0.21 μm, 0.22 μm, 0.23 μm, 0.24 μm, 0.25 μm, 0.26 μm, 0.27 μm,0.28 μm, 0.29 μm, 0.30 μm, 0.31 μm, 0.32 μm, 0.33 μm, 0.34 μm, 0.35 μm,0.36 μm, 0.37 μm, 0.38 μm, 0.39 μm, 0.40 μm, 0.41 μm, 0.42 μm, 0.43 μm,0.44 μm, 0.45 μm, 0.46 μm, 0.47 μm, 0.48 μm, 0.49 μm, 0.50 μm andlarger. The filters may be fabricated from any suitable non-reactivematerial including cellulose mixed ester (cellulose nitrate and acetate)(CME), polycarbonate (PC), polyvinylidene fluoride (PVDF),polyethersulfone (PES), polytetrafluoroethylene (PTFE), nylon, glassfiber, or metal substrates as in the case of laser or electrochemicaletching.

The length of the channel structure 402 may vary depending on the volumeof the cell culture to be grown and the optical density of the cellculture to be concentrated. The length of the channel structuretypically is from 60 mm to 300 mm, or from 70 mm to 200 mm, or from 80mm to 100 mm. The cross-section configuration of the flow channel 402may be round, elliptical, oval, square, rectangular, trapezoidal, orirregular. If square, rectangular, or another shape with generallystraight sides, the cross section may be from about 10 μm to 1000 μmwide, or from 200 μm to 800 μm wide, or from 300 μm to 700 μm wide, orfrom 400 μm to 600 μm wide; and from about 10 μm to 1000 μm high, orfrom 200 μm to 800 μm high, or from 300 μm to 700 μm high, or from 400μm to 600 μm high. If the cross section of the flow channel 102 isgenerally round, oval or elliptical, the radius of the channel may befrom about 50 μm to 1000 μm in hydraulic radius, or from 5 μm to 800 μmin hydraulic radius, or from 200 μm to 700 μm in hydraulic radius, orfrom 300 μm to 600 μm wide in hydraulic radius, or from about 200 to 500μm in hydraulic radius. Moreover, the volume of the channel in theretentate 422 and permeate 420 members may be different depending on thedepth of the channel in each member.

FIG. 4B shows front perspective (right) and rear perspective (left)views of a reservoir assembly 450 configured to be used with thetangential flow assembly 410 seen in FIG. 4A. Seen in the frontperspective view (e.g., “front” being the side of reservoir assembly 450that is coupled to the tangential flow assembly 410 seen in FIG. 4A) areretentate reservoirs 452 on either side of permeate reservoir 454. Alsoseen are permeate ports 426, retentate ports 428, and three threads ormating elements 425 for countersinks 423 (countersinks 423 not seen inthis FIG. 4B). Threads or mating elements 425 for countersinks 423 areconfigured to mate or couple the tangential flow assembly 410 (seen inFIG. 4A) to reservoir assembly 450. Alternatively or in addition,fasteners, sonic welding or heat stakes may be used to mate or couplethe tangential flow assembly 410 to reservoir assembly 450. In additionis seen gasket 445 covering the top of reservoir assembly 450. Gasket445 is described in detail in relation to FIG. 4E. At left in FIG. 4B isa rear perspective view of reservoir assembly 1250, where “rear” is theside of reservoir assembly 450 that is not coupled to the tangentialflow assembly. Seen are retentate reservoirs 452, permeate reservoir454, and gasket 445.

The TFF device (e.g., either the tangential flow filtration assembly,reservoir assembly, or both) may be fabricated from any robust materialin which channels (and channel branches) may be milled includingstainless steel, silicon, glass, aluminum, or plastics includingcyclic-olefin copolymer (COC), cyclo-olefin polymer (COP), polystyrene,polyvinyl chloride, polyethylene, polyamide, polyethylene,polypropylene, acrylonitrile butadiene, polycarbonate,polyetheretheketone (PEEK), poly(methyl methylacrylate) (PMMA),polysulfone, and polyurethane, and co-polymers of these and otherpolymers. If the TFF device/module is disposable, preferably it is madeof plastic. In some embodiments, the material used to fabricate the TFFdevice/module is thermally-conductive so that the cell culture may beheated or cooled to a desired temperature. In certain embodiments, theTFF device is formed by precision mechanical machining, laser machining,electro discharge machining (for metal devices); wet or dry etching (forsilicon devices); dry or wet etching, powder or sandblasting,photostructuring (for glass devices); or thermoforming, injectionmolding, hot embossing, or laser machining (for plastic devices) usingthe materials mentioned above that are amenable to this mass productiontechniques.

FIG. 4C depicts a top-down view of the reservoir assemblies 450 shown inFIG. 4B. FIG. 4D depicts a cover 444 for reservoir assembly 450 shown inFIGS. 4B and 4E depicts a gasket 445 that in operation is disposed oncover 444 of reservoir assemblies 450 shown in FIG. 4B. FIG. 4C is atop-down view of reservoir assembly 450, showing the tops of the tworetentate reservoirs 452, one on either side of permeate reservoir 454.Also seen are grooves 432 that will mate with a pneumatic port (notshown), and fluid channels 434 that reside at the bottom of retentatereservoirs 452, which fluidically couple the retentate reservoirs 452with the retentate ports 428 (not shown), via the through-holes for theretentate ports in permeate member 420 and membrane 424 (also notshown). FIG. 4D depicts a cover 444 that is configured to be disposedupon the top of reservoir assembly 450. Cover 444 has round cut-outs atthe top of retentate reservoirs 452 and permeate/filtrate reservoir 454.Again at the bottom of retentate reservoirs 452 fluid channels 434 canbe seen, where fluid channels 434 fluidically couple retentatereservoirs 452 with the retentate ports 428 (not shown). Also shown arethree pneumatic ports 430 for each retentate reservoir 452 andpermeate/filtrate reservoir 454. FIG. 4E depicts a gasket 445 that isconfigures to be disposed upon the cover 444 of reservoir assembly 450.Seen are three fluid transfer ports 442 for each retentate reservoir 452and for permeate/filtrate reservoir 454. Again, three pneumatic ports430, for each retentate reservoir 452 and for permeate/filtratereservoir 454, are shown.

The overall work flow for cell growth comprises loading a cell cultureto be grown into a first retentate reservoir, optionally bubbling air oran appropriate gas through the cell culture, passing or flowing the cellculture through the first retentate port then tangentially through theTFF channel structure while collecting medium or buffer through one orboth of the permeate ports 406, collecting the cell culture through asecond retentate port 404 into a second retentate reservoir, optionallyadding additional or different medium to the cell culture and optionallybubbling air or gas through the cell culture, then repeating theprocess, all while measuring, e.g., the optical density of the cellculture in the retentate reservoirs continuously or at desiredintervals. Measurements of optical densities (OD) at programmed timeintervals are accomplished using a 600 nm Light Emitting Diode (LED)that has been columnated through an optic into the retentatereservoir(s) containing the growing cells. The light continues through acollection optic to the detection system which consists of a (digital)gain-controlled silicone photodiode. Generally, optical density is shownas the absolute value of the logarithm with base 10 of the powertransmission factors of an optical attenuator: OD=−log 10 (Powerout/Power in). Since OD is the measure of optical attenuation—that is,the sum of absorption, scattering, and reflection—the TFF device ODmeasurement records the overall power transmission, so as the cells growand become denser in population, the OD (the loss of signal) increases.The OD system is pre-calibrated against OD standards with these valuesstored in an on-board memory accessible by the measurement program.

In the channel structure, the membrane bifurcating the flow channelsretains the cells on one side of the membrane (the retentate side 422)and allows unwanted medium or buffer to flow across the membrane into afiltrate or permeate side (e.g., permeate member 420) of the device.Bubbling air or other appropriate gas through the cell culture bothaerates and mixes the culture to enhance cell growth. During theprocess, medium that is removed during the flow through the channelstructure is removed through the permeate/filtrate ports 406.Alternatively, cells can be grown in one reservoir with bubbling oragitation without passing the cells through the TFF channel from onereservoir to the other.

The overall work flow for cell concentration using the TFF device/moduleinvolves flowing a cell culture or cell sample tangentially through thechannel structure. As with the cell growth process, the membranebifurcating the flow channels retains the cells on one side of themembrane and allows unwanted medium or buffer to flow across themembrane into a permeate/filtrate side (e.g., permeate member 420) ofthe device. In this process, a fixed volume of cells in medium or bufferis driven through the device until the cell sample is collected into oneof the retentate ports 404, and the medium/buffer that has passedthrough the membrane is collected through one or both of thepermeate/filtrate ports 406. All types of prokaryotic and eukaryoticcells—both adherent and non-adherent cells—can be grown in the TFFdevice. Adherent cells may be grown on beads or other cell scaffoldssuspended in medium that flow through the TFF device.

The medium or buffer used to suspend the cells in the cell concentrationdevice/module may be any suitable medium or buffer for the type of cellsbeing transformed or transfected, such as in this case bacteria cells;thus, LB or SOC media may be used, and the media may be provided in areagent cartridge as part of a kit.

In both the cell growth and concentration processes, passing the cellsample through the TFF device and collecting the cells in one of theretentate ports 404 while collecting the medium in one of thepermeate/filtrate ports 406 is considered “one pass” of the cell sample.The transfer between retentate reservoirs “flips” the culture. Theretentate and permeate ports collecting the cells and medium,respectively, for a given pass reside on the same end of TFFdevice/module with fluidic connections arranged so that there are twodistinct flow layers for the retentate and permeate/filtrate sides, butif the retentate port 404 resides on the retentate member ofdevice/module (that is, the cells are driven through the channel abovethe membrane and the filtrate (medium) passes to the portion of thechannel below the membrane), the permeate/filtrate port 406 will resideon the permeate member of device/module and vice versa (that is, if thecell sample is driven through the channel below the membrane, thefiltrate (medium) passes to the portion of the channel above themembrane). Due to the high pressures used to transfer the cell cultureand fluids through the flow channel of the TFF device, the effect ofgravity is negligible.

At the conclusion of a “pass” in either of the growth and concentrationprocesses, the cell sample is collected by passing through the retentateport 404 and into the retentate reservoir (not shown). To initiateanother “pass”, the cell sample is passed again through the TFF device,this time in a flow direction that is reversed from the first pass. Thecell sample is collected by passing through the retentate port 404 andinto retentate reservoir (not shown) on the opposite end of thedevice/module from the retentate port 404 that was used to collect cellsduring the first pass. Likewise, the medium/buffer that passes throughthe membrane on the second pass is collected through the permeate port406 on the opposite end of the device/module from the permeate port 406that was used to collect the filtrate during the first pass, or throughboth ports. This alternating process of passing the retentate (theconcentrated cell sample) through the device/module is repeated untilthe cells have been grown to a desired optical density, and/orconcentrated to a desired volume, and both permeate ports (i.e., ifthere are more than one) can be open during the passes to reduceoperating time. In addition, buffer exchange may be effected by adding adesired buffer (or fresh medium) to the cell sample in the retentatereservoir, before initiating another “pass”, and repeating this processuntil the old medium or buffer is diluted and filtered out and the cellsreside in fresh medium or buffer. Note that buffer exchange and cellgrowth may (and typically do) take place simultaneously, and bufferexchange and cell concentration may (and typically do) take placesimultaneously. For further information and alternative embodiments onTFFs see, e.g., U.S. Ser. No. 16/561,701, filed 5 Sep. 2019.

The Cell Transformation Module

FIG. 5A depicts an exemplary combination reagent cartridge andelectroporation device 500 (“cartridge”) that may be used in anautomated multi-module cell processing instrument along with the TFFmodule to effect efficient editing in bacteria. In addition, in certainembodiments the material used to fabricate the cartridge isthermally-conductive, as in certain embodiments the cartridge 500contacts a thermal device (not shown), such as a Peltier device orthermoelectric cooler, that heats or cools reagents in the reagentreservoirs or reservoirs 504. Reagent reservoirs or reservoirs 504 maybe reservoirs into which individual tubes of reagents are inserted asshown in FIG. 5A, or the reagent reservoirs may hold the reagentswithout inserted tubes. Additionally, the reservoirs in a reagentcartridge may be configured for any combination of tubes, co-joinedtubes, and direct-fill of reagents.

In one embodiment, the reagent reservoirs or reservoirs 504 of reagentcartridge 500 are configured to hold various size tubes, including,e.g., 250 ml tubes, 25 ml tubes, 10 ml tubes, 5 ml tubes, and Eppendorfor microcentrifuge tubes. In yet another embodiment, all reservoirs maybe configured to hold the same size tube, e.g., 5 ml tubes, andreservoir inserts may be used to accommodate smaller tubes in thereagent reservoir. In yet another embodiment—particularly in anembodiment where the reagent cartridge is disposable—the reagentreservoirs hold reagents without inserted tubes. In this disposableembodiment, the reagent cartridge may be part of a kit, where thereagent cartridge is pre-filled with reagents and the receptacles orreservoirs sealed with, e.g., foil, heat seal acrylic or the like andpresented to a consumer where the reagent cartridge can then be used inan automated multi-module cell processing instrument. As one of ordinaryskill in the art will appreciate given the present disclosure, thereagents contained in the reagent cartridge will vary depending on workflow; that is, the reagents will vary depending on the processes towhich the cells are subjected in the automated multi-module cellprocessing instrument, e.g., protein production, cell transformation andculture, cell editing, etc.

Reagents such as cell samples, enzymes, buffers, nucleic acid vectors,expression cassettes, proteins or peptides, reaction components (suchas, e.g., MgCl₂, dNTPs, nucleic acid assembly reagents, gap repairreagents, and the like), wash solutions, ethanol, and magnetic beads fornucleic acid purification and isolation, etc. may be positioned in thereagent cartridge at a known position. In some embodiments of cartridge500, the cartridge comprises a script (not shown) readable by aprocessor (not shown) for dispensing the reagents. Also, the cartridge500 as one component in an automated multi-module cell processinginstrument may comprise a script specifying two, three, four, five, tenor more processes to be performed by the automated multi-module cellprocessing instrument. In certain embodiments, the reagent cartridge isdisposable and is pre-packaged with reagents tailored to performingspecific cell processing protocols, e.g., genome editing or proteinproduction. Because the reagent cartridge contents vary whilecomponents/modules of the automated multi-module cell processinginstrument or system may not, the script associated with a particularreagent cartridge matches the reagents used and cell processesperformed. Thus, e.g., reagent cartridges may be pre-packaged withreagents for genome editing and a script that specifies the processsteps for performing genome editing in an automated multi-module cellprocessing instrument, or, e.g., reagents for protein expression and ascript that specifies the process steps for performing proteinexpression in an automated multi-module cell processing instrument.

For example, the reagent cartridge may comprise a script to pipettecompetent cells from a reservoir, transfer the cells to a transformationmodule, pipette a nucleic acid solution comprising a vector withexpression cassette from another reservoir in the reagent cartridge,transfer the nucleic acid solution to the transformation module,initiate the transformation process for a specified time, then move thetransformed cells to yet another reservoir in the reagent cassette or toanother module such as a cell growth module in the automatedmulti-module cell processing instrument. In another example, the reagentcartridge may comprise a script to transfer a nucleic acid solutioncomprising a vector from a reservoir in the reagent cassette, nucleicacid solution comprising editing oligonucleotide cassettes in areservoir in the reagent cassette, and a nucleic acid assembly mix fromanother reservoir to the nucleic acid assembly/desalting module, ifpresent. The script may also specify process steps performed by othermodules in the automated multi-module cell processing instrument. Forexample, the script may specify that the nucleic acid assembly/desaltingreservoir be heated to 50° C. for 30 min to generate an assembledproduct; and desalting and resuspension of the assembled product viamagnetic bead-based nucleic acid purification involving a series ofpipette transfers and mixing of magnetic beads, ethanol wash, andbuffer.

As described in relation to FIGS. 5B and 5C below, the exemplary reagentcartridges for use in the automated multi-module cell processinginstruments may include one or more electroporation devices, preferablyflow-through electroporation (FTEP) devices. In yet other embodiments,the reagent cartridge is separate from the transformation module.Electroporation is a widely-used method for permeabilization of cellmembranes that works by temporarily generating pores in the cellmembranes with electrical stimulation. Applications of electroporationinclude the delivery of DNA, RNA, siRNA, peptides, proteins, antibodies,drugs or other substances to a variety of cells such as mammalian cells(including human cells), plant cells, archea, yeasts, other eukaryoticcells, bacteria, and other cell types. Electrical stimulation may alsobe used for cell fusion in the production of hybridomas or other fusedcells. During a typical electroporation procedure, cells are suspendedin a buffer or medium that is favorable for cell survival. For bacterialcell electroporation, low conductance mediums, such as water, glycerolsolutions and the like, are often used to reduce the heat production bytransient high current. In traditional electroporation devices, thecells and material to be electroporated into the cells (collectively“the cell sample”) are placed in a cuvette embedded with two flatelectrodes for electrical discharge. For example, Bio-Rad (Hercules,Calif.) makes the GENE PULSER XCELL™ line of products to electroporatecells in cuvettes. Traditionally, electroporation requires high fieldstrength; however, the flow-through electroporation devices included inthe reagent cartridges achieve high efficiency cell electroporation withlow toxicity. The reagent cartridges of the disclosure allow forparticularly easy integration with robotic liquid handlinginstrumentation that is typically used in automated instruments andsystems such as air displacement pipettors. Such automatedinstrumentation includes, but is not limited to, off-the-shelf automatedliquid handling systems from Tecan (Mannedorf, Switzerland), Hamilton(Reno, Nev.), Beckman Coulter (Fort Collins, Colo.), etc.

FIGS. 5B and 5C are top perspective and bottom perspective views,respectively, of an exemplary FTEP device 550 that may be part of (e.g.,a component in) reagent cartridge 500 in FIG. 5A or may be a stand-alonemodule; that is, not a part of a reagent cartridge or other module. FIG.5B depicts an FTEP device 550. The FTEP device 550 has wells that definecell sample inlets 552 and cell sample outlets 554. FIG. 5C is a bottomperspective view of the FTEP device 550 of FIG. 5B. An inlet well 552and an outlet well 554 can be seen in this view. Also seen in FIG. 5Care the bottom of an inlet 562 corresponding to well 552, the bottom ofan outlet 564 corresponding to the outlet well 554, the bottom of adefined flow channel 566 and the bottom of two electrodes 568 on eitherside of flow channel 566. The FTEP devices may comprise push-pullpneumatic means to allow multi-pass electroporation procedures; that is,cells to electroporated may be “pulled” from the inlet toward the outletfor one pass of electroporation, then be “pushed” from the outlet end ofthe FTEP device toward the inlet end to pass between the electrodesagain for another pass of electroporation. Further, this process may berepeated one to many times. For additional information regarding FTEPdevices, see, e.g., U.S. Pat. No. 10,435,713, issued 8 Oct. 2019; U.S.Pat. No. 10,443,074, issued 15 Oct. 2019; U.S. Pat. No. 10,415,258,issued 17 Sep. 2019; and U.S. Pat. No. 10,323,258, issued 18 Jun. 2019.Further, other embodiments of the reagent cartridge may provide oraccommodate electroporation devices that are not configured as FTEPdevices, such as those described in U.S. Ser. No. 16/109,156, filed 22Aug. 2018. For reagent cartridges useful in the present automatedmulti-module cell processing instruments, see, e.g., U.S. Pat. No.10,376,889, issued 13 Aug. 2019; and U.S. Ser. No. 16,451,601, filed 25Jun. 2019.

Additional details of the FTEP devices are illustrated in FIGS. 5D-5F.Note that in the FTEP devices in FIGS. 5D-5F the electrodes are placedsuch that a first electrode is placed between an inlet and a narrowedregion of the flow channel, and the second electrode is placed betweenthe narrowed region of the flow channel and an outlet. FIG. 5D shows atop planar view of an FTEP device 550 having an inlet 552 forintroducing a fluid containing cells and exogenous material into FTEPdevice 550 and an outlet 554 for removing the transformed cells from theFTEP following electroporation. The electrodes 568 are introducedthrough channels (not shown) in the device. FIG. 5E shows a cutaway viewfrom the top of the FTEP device 550, with the inlet 552, outlet 554, andelectrodes 568 positioned with respect to a flow channel 566. FIG. 5Fshows a side cutaway view of FTEP device 550 with the inlet 552 andinlet channel 572, and outlet 554 and outlet channel 574. The electrodes568 are positioned in electrode channels 576 so that they are in fluidcommunication with the flow channel 566, but not directly in the path ofthe cells traveling through the flow channel 566. Note that the firstelectrode is placed between the inlet and the narrowed region of theflow channel, and the second electrode is placed between the narrowedregion of the flow channel and the outlet. The electrodes 568 in thisaspect of the device are positioned in the electrode channels 576 whichare generally perpendicular to the flow channel 566 such that the fluidcontaining the cells and exogenous material flows from the inlet channel572 through the flow channel 566 to the outlet channel 574, and in theprocess fluid flows into the electrode channels 376 to be in contactwith the electrodes 568. In this aspect, the inlet channel, outletchannel and electrode channels all originate from the same planar sideof the device. In certain aspects, however, the electrodes may beintroduced from a different planar side of the FTEP device than theinlet and outlet channels.

In the FTEP devices of the disclosure, the toxicity level of thetransformation results in greater than 30% viable cells afterelectroporation, preferably greater than 35%, 40%, 45%, 50%, 55%, 60%,70%, 75%, 80%, 85%, 90%, 95% or even 99% viable cells followingtransformation, depending on the cell type and the nucleic acids beingintroduced into the cells.

The housing of the FTEP device can be made from many materials dependingon whether the FTEP device is to be reused, autoclaved, or isdisposable, including stainless steel, silicon, glass, resin, polyvinylchloride, polyethylene, polyamide, polystyrene, polyethylene,polypropylene, acrylonitrile butadiene, polycarbonate,polyetheretheketone (PEEK), polysulfone and polyurethane, co-polymers ofthese and other polymers. Similarly, the walls of the channels in thedevice can be made of any suitable material including silicone, resin,glass, glass fiber, polyvinyl chloride, polyethylene, polyamide,polyethylene, polypropylene, acrylonitrile butadiene, polycarbonate,polyetheretheketone (PEEK), polysulfone and polyurethane, co-polymers ofthese and other polymers. Preferred materials include crystal styrene,cyclo-olefin polymer (COP) and cyclic olephin co-polymers (COC), whichallow the device to be formed entirely by injection molding in one piecewith the exception of the electrodes and, e.g., a bottom sealing film ifpresent.

The FTEP devices described herein (or portions of the FTEP devices) canbe created or fabricated via various techniques, e.g., as entire devicesor by creation of structural layers that are fused or otherwise coupled.For example, for metal FTEP devices, fabrication may include precisionmechanical machining or laser machining; for silicon FTEP devices,fabrication may include dry or wet etching; for glass FTEP devices,fabrication may include dry or wet etching, powder blasting,sandblasting, or photostructuring; and for plastic FTEP devicesfabrication may include thermoforming, injection molding, hot embossing,or laser machining. The components of the FTEP devices may bemanufactured separately and then assembled, or certain components of theFTEP devices (or even the entire FTEP device except for the electrodes)may be manufactured (e.g., using 3D printing) or molded (e.g., usinginjection molding) as a single entity, with other components added aftermolding. For example, housing and channels may be manufactured or moldedas a single entity, with the electrodes later added to form the FTEPunit. Alternatively, the FTEP device may also be formed in two or moreparallel layers, e.g., a layer with the horizontal channel and filter, alayer with the vertical channels, and a layer with the inlet and outletports, which are manufactured and/or molded individually and assembledfollowing manufacture.

In specific aspects, the FTEP device can be manufactured using a circuitboard as a base, with the electrodes, filter and/or the flow channelformed in the desired configuration on the circuit board, and theremaining housing of the device containing, e.g., the one or more inletand outlet channels and/or the flow channel formed as a separate layerthat is then sealed onto the circuit board. The sealing of the top ofthe housing onto the circuit board provides the desired configuration ofthe different elements of the FTEP devices of the disclosure. Also, twoto many FTEP devices may be manufactured on a single substrate, thenseparated from one another thereafter or used in parallel. In certainembodiments, the FTEP devices are reusable and, in some embodiments, theFTEP devices are disposable. In additional embodiments, the FTEP devicesmay be autoclavable.

The electrodes 408 can be formed from any suitable metal, such ascopper, stainless steel, titanium, aluminum, brass, silver, rhodium,gold or platinum, or graphite. One preferred electrode material is alloy303 (UNS330300) austenitic stainless steel. An applied electric fieldcan destroy electrodes made from of metals like aluminum. If amultiple-use (i.e., non-disposable) flow-through FTEP device isdesired—as opposed to a disposable, one-use flow-through FTEP device—theelectrode plates can be coated with metals resistant to electrochemicalcorrosion. Conductive coatings like noble metals, e.g., gold, can beused to protect the electrode plates.

As mentioned, the FTEP devices may comprise push-pull pneumatic means toallow multi-pass electroporation procedures; that is, cells toelectroporated may be “pulled” from the inlet toward the outlet for onepass of electroporation, then be “pushed” from the outlet end of theflow-through FTEP device toward the inlet end to pass between theelectrodes again for another pass of electroporation. This process maybe repeated one to many times.

Depending on the type of cells to be electroporated (e.g., bacterial,yeast, mammalian) and the configuration of the electrodes, the distancebetween the electrodes in the flow channel can vary widely. For example,where the flow channel decreases in width, the flow channel may narrowto between 10 μm and 5 mm, or between 25 μm and 3 mm, or between 50 μmand 2 mm, or between 75 μm and 1 mm. The distance between the electrodesin the flow channel may be between 1 mm and 10 mm, or between 2 mm and 8mm, or between 3 mm and 7 mm, or between 4 mm and 6 mm. The overall sizeof the FTEP device may be from 3 cm to 15 cm in length, or 4 cm to 12 cmin length, or 4.5 cm to 10 cm in length. The overall width of the FTEPdevice may be from 0.5 cm to 5 cm, or from 0.75 cm to 3 cm, or from 1 cmto 2.5 cm, or from 1 cm to 1.5 cm.

The region of the flow channel that is narrowed is wide enough so thatat least two cells can fit in the narrowed portion side-by-side. Forexample, a typical bacterial cell is 1 μm in diameter; thus, thenarrowed portion of the flow channel of the FTEP device used totransform such bacterial cells will be at least 2 μm wide. In anotherexample, if a mammalian cell is approximately 50 μm in diameter, thenarrowed portion of the flow channel of the FTEP device used totransform such mammalian cells will be at least 100 μm wide. That is,the narrowed portion of the FTEP device will not physically contort or“squeeze” or occlude the cells being transformed.

In embodiments of the FTEP device where reservoirs are used to introducecells and exogenous material into the FTEP device, the reservoirs rangein volume from 100 μL to 10 mL, or from 500 μL to 75 mL, or from 1 mL to5 mL. The flow rate in the FTEP ranges from 0.1 mL to 5 mL per minute,or from 0.5 mL to 3 mL per minute, or from 1.0 mL to 2.5 mL per minute.The pressure in the FTEP device ranges from 1-30 psi, or from 2-10 psi,or from 3-5 psi.

To avoid different field intensities between the electrodes, theelectrodes should be arranged in parallel. Furthermore, the surface ofthe electrodes should be as smooth as possible without pin holes orpeaks. Electrodes having a roughness Rz of 1 to 10 μm are preferred. Inanother embodiment of the invention, the flow-through electroporationdevice comprises at least one additional electrode which applies aground potential to the FTEP device.

Cell Singulation and Enrichment Device

A module useful for performing the bacterial recovery, selection andediting methods depicted in FIG. 1A is a solid wall isolation,incubation, and normalization (SWIIN) module. FIG. 6A depicts anembodiment of a SWIIN module 650 from an exploded top perspective view.In SWIIN module 650 the retentate member is formed on the bottom of atop of a SWIIN module component and the permeate member is formed on thetop of the bottom of a SWIIN module component.

The SWIIN module 650 in FIG. 6A comprises from the top down, a reservoirgasket or cover 658, a retentate member 604 (where a retentate flowchannel cannot be seen in this FIG. 6A), a perforated member 601 swagedwith a filter (filter not seen in FIG. 6A), a permeate member 608comprising integrated reservoirs (permeate reservoirs 652 and retentatereservoirs 654), and two reservoir seals 662, which seal the bottom ofpermeate reservoirs 652 and retentate reservoirs 654. A permeate channel660 a can be seen disposed on the top of permeate member 608, defined bya raised portion 676 of serpentine channel 660 a, and ultrasonic tabs664 can be seen disposed on the top of permeate member 608 as well. Theperforations that form the wells on perforated member 601 are not seenin this FIG. 6A; however, through-holes 666 to accommodate theultrasonic tabs 664 are seen. In addition, supports 670 are disposed ateither end of SWIIN module 650 to support SWIIN module 650 and toelevate permeate member 608 and retentate member 604 above reservoirs652 and 654 to minimize bubbles or air entering the fluid path from thepermeate reservoir to serpentine channel 660 a or the fluid path fromthe retentate reservoir to serpentine channel 660 b (neither fluid pathis seen in this FIG. 6A).

In this FIG. 6A, it can be seen that the serpentine channel 660 a thatis disposed on the top of permeate member 608 traverses permeate member608 for most of the length of permeate member 608 except for the portionof permeate member 608 that comprises permeate reservoirs 652 andretentate reservoirs 654 and for most of the width of permeate member608. As used herein with respect to the distribution channels in theretentate member or permeate member, “most of the length” means about95% of the length of the retentate member or permeate member, or about90%, 85%, 80%, 75%, or 70% of the length of the retentate member orpermeate member. As used herein with respect to the distributionchannels in the retentate member or permeate member, “most of the width”means about 95% of the width of the retentate member or permeate member,or about 90%, 85%, 80%, 75%, or 70% of the width of the retentate memberor permeate member.

In this embodiment of a SWIIN module, the perforated member includesthrough-holes to accommodate ultrasonic tabs disposed on the permeatemember. Thus, in this embodiment the perforated member is fabricatedfrom 316 stainless steel, and the perforations form the walls ofmicrowells while a filter or membrane is used to form the bottom of themicrowells. Typically, the perforations (microwells) are approximately150 μm-200 μm in diameter, and the perforated member is approximately125 μm deep, resulting in microwells having a volume of approximately2.5 nl, with a total of approximately 200,000 microwells. The distancebetween the microwells is approximately 279 μm center-to-center. Thoughhere the microwells have a volume of approximately 2.5 nl, the volume ofthe microwells may be from 1 to 25 nl, or preferably from 2 to 10 nl,and even more preferably from 2 to 4 nl. As for the filter or membrane,like the filter described previously, filters appropriate for use aresolvent resistant, contamination free during filtration, and are able toretain the types and sizes of cells of interest. For example, in orderto retain small cell types such as bacterial cells, pore sizes can be aslow as 0.10 μm, however for other cell types (e.g., such as formammalian cells), the pore sizes can be as high as 10.0 μm-20.0 μm ormore. Indeed, the pore sizes useful in the cell concentrationdevice/module include filters with sizes from 0.10 μm, 0.11 μm, 0.12 μm,0.13 μm, 0.14 μm, 0.15 μm, 0.16 μm, 0.17 μm, 0.18 μm, 0.19 μm, 0.20 μm,0.21 μm, 0.22 μm, 0.23 μm, 0.24 μm, 0.25 μm, 0.26 μm, 0.27 μm, 0.28 μm,0.29 μm, 0.30 μm, 0.31 μm, 0.32 μm, 0.33 μm, 0.34 μm, 0.35 μm, 0.36 μm,0.37 μm, 0.38 μm, 0.39 μm, 0.40 μm, 0.41 μm, 0.42 μm, 0.43 μm, 0.44 μm,0.45 μm, 0.46 μm, 0.47 μm, 0.48 μm, 0.49 μm, 0.50 μm and larger. Thefilters may be fabricated from any suitable material including cellulosemixed ester (cellulose nitrate and acetate) (CME), polycarbonate (PC),polyvinylidene fluoride (PVDF), polyethersulfone (PES),polytetrafluoroethylene (PTFE), nylon, or glass fiber.

The cross-section configuration of the mated serpentine channel may beround, elliptical, oval, square, rectangular, trapezoidal, or irregular.If square, rectangular, or another shape with generally straight sides,the cross section may be from about 2 mm to 15 mm wide, or from 3 mm to12 mm wide, or from 5 mm to 10 mm wide. If the cross section of themated serpentine channel is generally round, oval or elliptical, theradius of the channel may be from about 3 mm to 20 mm in hydraulicradius, or from 5 mm to 15 mm in hydraulic radius, or from 8 mm to 12 mmin hydraulic radius.

Serpentine channels 660 a and 660 b can have approximately the samevolume or a different volume. For example, each “side” or portion 660 a,660 b of the serpentine channel may have a volume of, e.g., 2 mL, orserpentine channel 660 a of permeate member 608 may have a volume of 2mL, and the serpentine channel 660 b of retentate member 604 may have avolume of, e.g., 3 mL. The volume of fluid in the serpentine channel mayrange from about 2 mL to about 80 mL, or about 4 mL to 60 mL, or from 5mL to 40 mL, or from 6 mL to 20 mL (note these volumes apply to a SWIINmodule comprising a, e.g., 50-500K perforation member). The volume ofthe reservoirs may range from 5 mL to 50 mL, or from 7 mL to 40 mL, orfrom 8 mL to 30 mL or from 10 mL to 20 mL, and the volumes of allreservoirs may be the same or the volumes of the reservoirs may differ(e.g., the volume of the permeate reservoirs is greater than that of theretentate reservoirs).

The serpentine channel portions 660 a and 660 b of the permeate member608 and retentate member 604, respectively, are approximately 200 mmlong, 130 mm wide, and 4 mm thick, though in other embodiments, theretentate and permeate members can be from 75 mm to 400 mm in length, orfrom 100 mm to 300 mm in length, or from 150 mm to 250 mm in length;from 50 mm to 250 mm in width, or from 75 mm to 200 mm in width, or from100 mm to 150 mm in width; and from 2 mm to 15 mm in thickness, or from4 mm to 10 mm in thickness, or from 5 mm to 8 mm in thickness.Embodiments the retentate (and permeate) members may be fabricated fromPMMA (poly(methyl methacrylate) or other materials may be used,including polycarbonate, cyclic olefin co-polymer (COC), glass,polyvinyl chloride, polyethylene, polyamide, polypropylene, polysulfone,polyurethane, and co-polymers of these and other polymers. Preferably atleast the retentate member is fabricated from a transparent material sothat the cells can be visualized (see, e.g., FIG. 6E and the descriptionthereof). For example, a video camera may be used to monitor cell growthby, e.g., density change measurements based on an image of an emptywell, with phase contrast, or if, e.g., a chromogenic marker, such as achromogenic protein, is used to add a distinguishable color to thecells. Chromogenic markers such as blitzen blue, dreidel teal, virginiaviolet, vixen purple, prancer purple, tinsel purple, maccabee purple,donner magenta, cupid pink, seraphina pink, scrooge orange, and leororange (the Chromogenic Protein Paintbox, all available from ATUM(Newark, Calif.)) obviate the need to use fluorescence, althoughfluorescent cell markers, fluorescent proteins, and chemiluminescentcell markers may also be used.

Because the retentate member preferably is transparent, colony growth inthe SWIIN module can be monitored by automated devices such as thosesold by JoVE (ScanLag™ system, Cambridge, Mass.) (also seeLevin-Reisman, et al., Nature Methods, 7:737-39 (2010)). Cell growthfor, e.g., mammalian cells may be monitored by, e.g., the growth monitorsold by IncuCyte (Ann Arbor, Mich.) (see also, Choudhry, PLos One,11(2):e0148469 (2016)). Further, automated colony pickers may beemployed, such as those sold by, e.g., TECAN (Pickolo™ system,Mannedorf, Switzerland); Hudson Inc. (RapidPick™, Springfield, N.J.);Molecular Devices (QPix 400™ system, San Jose, Calif.); and SingerInstruments (PIXL™ system, Somerset, UK).

Due to the heating and cooling of the SWIIN module, condensation mayaccumulate on the retentate member which may interfere with accuratevisualization of the growing cell colonies. Condensation of the SWIINmodule 650 may be controlled by, e.g., moving heated air over the top of(e.g., retentate member) of the SWIIN module 650, or by applying atransparent heated lid over at least the serpentine channel portion 660b of the retentate member 604. See, e.g., FIG. 6F and the descriptionthereof infra.

In SWIIN module 650 cells and medium—at a dilution appropriate forPoisson or substantial Poisson distribution of the cells in themicrowells of the perforated member—are flowed into serpentine channel660 b from ports in retentate member 604, and the cells settle in themicrowells while the medium passes through the filter into serpentinechannel 660 a in permeate member 608. The cells are retained in themicrowells of perforated member 601 as the cells cannot travel throughfilter 603. Appropriate medium may be introduced into permeate member608 through permeate ports 611. The medium flows upward through filter603 to nourish the cells in the microwells (perforations) of perforatedmember 601. Additionally, buffer exchange can be effected by cyclingmedium through the retentate and permeate members. In operation, thecells are deposited into the microwells, are grown for an initial, e.g.,2-100 doublings, editing is induced by, e.g., raising the temperature ofthe SWIIN to 42° C. to induce a temperature inducible promoter or byremoving growth medium from the permeate member and replacing the growthmedium with a medium comprising a chemical component that induces aninducible promoter.

Once editing has taken place, the temperature of the SWIIN may bedecreased, or the inducing medium may be removed and replaced with freshmedium lacking the chemical component thereby de-activating theinducible promoter. The cells then continue to grow in the SWIIN module650 until the growth of the cell colonies in the microwells isnormalized. For the normalization protocol, once the colonies arenormalized, the colonies are flushed from the microwells by applyingfluid or air pressure (or both) to the permeate member serpentinechannel 660 a and thus to filter 603 and pooled. Alternatively, ifcherry picking is desired, the growth of the cell colonies in themicrowells is monitored, and slow-growing colonies are directlyselected; or, fast-growing colonies are eliminated.

FIG. 6B is a top perspective view of a SWIIN module with the retentateand perforated members in partial cross section. In this FIG. 6C, it canbe seen that serpentine channel 660 a is disposed on the top of permeatemember 608 is defined by raised portions 676 and traverses permeatemember 608 for most of the length and width of permeate member 608except for the portion of permeate member 608 that comprises thepermeate and retentate reservoirs (note only one retentate reservoir 652can be seen). Moving from left to right, reservoir gasket 658 isdisposed upon the integrated reservoir cover 678 (cover not seen in thisFIG. 6C) of retentate member 604. Gasket 658 comprises reservoir accessapertures 632 a, 632 b, 632 c, and 632 d, as well as pneumatic ports 633a, 633 b, 633 c and 633 d. Also at the far left end is support 670.Disposed under permeate reservoir 652 can be seen one of two reservoirseals 662. In addition to the retentate member being in cross section,the perforated member 601 and filter 603 (filter 603 is not seen in thisFIG. 6C) are in cross section. Note that there are a number ofultrasonic tabs 664 disposed at the right end of SWIIN module 650 and onraised portion 676 which defines the channel turns of serpentine channel660 a, including ultrasonic tabs 664 extending through through-holes 666of perforated member 601. There is also a support 670 at the end distalreservoirs 652, 654 of permeate member 608.

FIG. 6C is a side perspective view of an assembled SWIIIN module 650,including, from right to left, reservoir gasket 658 disposed uponintegrated reservoir cover 678 (not seen) of retentate member 604.Gasket 658 may be fabricated from rubber, silicone, nitrile rubber,polytetrafluoroethylene, a plastic polymer such aspolychlorotrifluoroethylene, or other flexible, compressible material.Gasket 658 comprises reservoir access apertures 632 a, 632 b, 632 c, and632 d, as well as pneumatic ports 633 a, 633 b, 633 c and 633 d. Also atthe far-left end is support 670 of permeate member 608. In addition,permeate reservoir 652 can be seen, as well as one reservoir seal 662.At the far-right end is a second support 670.

Imaging of cell colonies growing in the wells of the SWIIN is desired inmost implementations for, e.g., monitoring both cell growth and deviceperformance and imaging is necessary for cherry-picking implementations.Real-time monitoring of cell growth in the SWIIN requires backlighting,retentate plate (top plate) condensation management and a system-levelapproach to temperature control, air flow, and thermal management. Insome implementations, imaging employs a camera or CCD device withsufficient resolution to be able to image individual wells. For example,in some configurations a camera with a 9-pixel pitch is used (that is,there are 9 pixels center-to-center for each well). Processing theimages may, in some implementations, utilize reading the images ingrayscale, rating each pixel from low to high, where wells with no cellswill be brightest (due to full or nearly-full light transmission fromthe backlight) and wells with cells will be dim (due to cells blockinglight transmission from the backlight). After processing the images,thresholding is performed to determine which pixels will be called“bright” or “dim”, spot finding is performed to find bright pixels andarrange them into blocks, and then the spots are arranged on a hexagonalgrid of pixels that correspond to the spots. Once arranged, the measureof intensity of each well is extracted, by, e.g., looking at one or morepixels in the middle of the spot, looking at several to many pixels atrandom or pre-set positions, or averaging X number of pixels in thespot. In addition, background intensity may be subtracted. Thresholdingis again used to call each well positive (e.g., containing cells) ornegative (e.g., no cells in the well). The imaging information may beused in several ways, including taking images at time points formonitoring cell growth. Monitoring cell growth can be used to, e.g.,remove the “muffin tops” of fast-growing cells followed by removal ofall cells or removal of cells in “rounds” as described above, or recovercells from specific wells (e.g., slow-growing cell colonies);alternatively, wells containing fast-growing cells can be identified andareas of UV light covering the fast-growing cell colonies can beprojected (or rastered with shutters) onto the SWIIN to irradiate orinhibit growth of those cells. Imaging may also be used to assure properfluid flow in the serpentine channel 660.

FIG. 6D depicts the embodiment of the SWIIN module in FIGS. 6A-6Cfurther comprising a heat management system including a heater and aheated cover. The heater cover facilitates the condensation managementthat is required for imaging. Assembly 698 comprises a SWIIN module 650seen lengthwise in cross section, where one permeate reservoir 652 isseen. Disposed immediately upon SWIIN module 650 is cover 694 anddisposed immediately below SWIIN module 650 is backlight 680, whichallows for imaging. Beneath and adjacent to the backlight and SWIINmodule is insulation 682, which is disposed over a heatsink 684. In thisFIG. 6E, the fins of the heatsink would be in-out of the page. Inaddition there is also axial fan 686 and heat sink 688, as well as twothermoelectric coolers 692, and a controller 690 to control thepneumatics, thermoelectric coolers, fan, solenoid valves, etc. Thearrows denote cool air coming into the unit and hot air being removedfrom the unit. It should be noted that control of heating allows forgrowth of many different types of cells (prokaryotic and eukaryotic) aswell as strains of cells that are, e.g., temperature sensitive, etc.,and allows use of temperature-sensitive promoters. Temperature controlallows for protocols to be adjusted to account for differences intransformation efficiency, cell growth and viability. For more detailsregarding solid wall isolation incubation and normalization devices seeU.S. Ser. No. 16/399,988, filed 30 Apr. 2019; Ser. No. 16/454,865, filed26 Jun. 2019; and Ser. No. 16/540,606, filed 14 Aug. 2019. Foralternative isolation, incubation and normalization modules, see U.S.Ser. No. 16/536,049, filed 8 Aug. 2019.

It should be apparent to one of ordinary skill in the art given thepresent disclosure that the process described may be recursive andmultiplexed; that is, cells may go through an editing workflow, then theresulting edited culture may go through another (or several or many)rounds of additional editing (e.g., recursive editing) with differentediting vectors. For example, the cells from round 1 of editing may bediluted and an aliquot of the edited cells edited by editing vector Amay be combined with editing vector B, an aliquot of the edited cellsedited by editing vector A may be combined with editing vector C, analiquot of the edited cells edited by editing vector A may be combinedwith editing vector D, and so on for a second round of editing. Afterround two, an aliquot of each of the double-edited cells may besubjected to a third round of editing, where, e.g., aliquots of each ofthe AB-, AC-, AD-edited cells are combined with additional editingvectors, such as editing vectors X, Y, and Z. That is that double-editedcells AB may be combined with and edited by vectors X, Y, and Z toproduce triple-edited edited cells ABX, ABY, and ABZ; double-editedcells AC may be combined with and edited by vectors X, Y, and Z toproduce triple-edited cells ACX, ACY, and ACZ; and double-edited cellsAD may be combined with and edited by vectors X, Y, and Z to producetriple-edited cells ADX, ADY, and ADZ, and so on. In this process, manypermutations and combinations of edits can be executed, leading to verydiverse cell populations and cell libraries. In any recursive process,it is advantageous to “cure” the previous engine and editing vectors (orsingle engine+editing vector in a single vector system). “Curing” is aprocess in which one or more vectors used in the prior round of editingis eliminated from the transformed cells.

Curing can be accomplished by, e.g., cleaving the vector(s) using acuring plasmid thereby rendering the editing and/or engine vector (orsingle, combined engine/editing vector) nonfunctional; diluting thevector(s) in the cell population via cell growth (that is, the moregrowth cycles the cells go through, the fewer daughter cells will retainthe editing or engine vector(s)), or by, e.g., utilizing aheat-sensitive origin of replication on the editing or engine vector (orcombined engine+editing vector). The conditions for curing will dependon the mechanism used for curing; that is, in this example, how thecuring plasmid cleaves the editing and/or engine vector.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent or imply that the experiments below are all of orthe only experiments performed. It will be appreciated by personsskilled in the art that numerous variations and/or modifications may bemade to the invention as shown in the specific aspects without departingfrom the spirit or scope of the invention as broadly described. Thepresent aspects are, therefore, to be considered in all respects asillustrative and not restrictive.

Example I: Editing Cassette and Backbone Amplification and Assembly

Editing Cassette Preparation: 5 nM oligonucleotides synthesized on achip were amplified using Q5 polymerase in 50 μL volumes. The PCRconditions were 95° C. for 1 minute; 8 rounds of 95° C. for 30seconds/60° C. for 30 seconds/72° C. for 2.5 minutes; with a final holdat 72° C. for 5 minutes. Following amplification, the PCR products weresubjected to SPRI cleanup, where 30 μL SPRI mix was added to the 50 μLPCR reactions and incubated for 2 minutes. The tubes were subjected to amagnetic field for 2 minutes, the liquid was removed, and the beads werewashed 2× with 80% ethanol, allowing 1 minute between washes. After thefinal wash, the beads were allowed to dry for 2 minutes, 50 μL 0.5×TE pH8.0 was added to the tubes, and the beads were vortexed to mix. Theslurry was incubated at room temperature for 2 minutes, then subjectedto the magnetic field for 2 minutes. The eluate was removed and the DNAquantified.

Following quantification, a second amplification procedure was carriedout using a dilution of the eluate from the SPRI cleanup. PCR wasperformed under the following conditions: 95° C. for 1 minute; 18 roundsof 95° C. for 30 seconds/72° C. for 2.5 minutes; with a final hold at72° C. for 5 minutes. Amplicons were checked on a 2% agarose gel andpools with the cleanest output(s) were identified. Amplificationproducts appearing to have heterodimers or chimeras were not used.

Backbone Preparation: A 10-fold serial dilution series of purifiedbackbone was performed, and each of the diluted backbone series wasamplified under the following conditions: 95° C. for 1 minute; then 30rounds of 95° C. for 30 seconds/60° C. for 1.5 minutes/72° C. for 2.5minutes; with a final hold at 72° C. for 5 minutes. After amplification,the amplified backbone was subjected to SPRI cleanup as described abovein relation to the cassettes. The backbone was eluted into 100 μL ddH₂Oand quantified before nucleic acid assembly.

Isothermal Nucleic Acid Assembly: 150 ng backbone DNA was combined with100 ng cassette DNA. An equal volume of 2× Gibson Master Mix was added,and the reaction was incubated for 45 minutes at 50° C. After assembly,the assembled backbone and cassettes were subjected to SPRI cleanup, asdescribed above.

Example II: Preparation of Competent Cells

A 1 mL aliquot of a freshly-grown overnight culture of MG1655 strain E.coli cells transformed with the engine vector was added to a 250 mLflask containing 100 mL LB/SOB+25 μg/mL chlor medium. The cells weregrown to 0.4-0.7 OD, and cell growth was halted by transferring theculture to ice for 10 minutes. The cells were pelleted at 8000×g in aJA-18 rotor for 5 minutes, washed 3× with 50 mL ice cold ddH₂O or 10%glycerol, and pelleted at 8000×g in JA-18 rotor for 5 minutes. Thewashed cells were resuspended in 5 mL ice cold 10% glycerol andaliquoted into 200 μL portions. Optionally at this point the glycerolstocks could be stored at −80° C. for later use.

Example III: Creation of New Cell Lines Transformed with Engine Vectors

Transformation: 1 μL of each of the different engine vector DNA (e.g.,one of the p197, p-197-recA or p197-srpRrecA engine vector) was added to50 μL MG1655 strain E. coli cells. The transformed cells were plated onLB plates with 25 μg/mL chloramphenicol (chlor) and incubated overnightto accumulate clonal isolates. The next day, a colony was picked, grownovernight in LB+25 μg/mL chlor, and glycerol stocks were prepared fromthe saturated overnight culture by adding 500 μL 50% glycerol to 1000 μLculture. The stocks of E. coli cells comprising the different enginevectors were frozen at −80° C.

Example IV: Transformation of Editing Vector (Triple Edit) into E cloni®

Transformation: 20 μL of the prepared editing vector Gibson Assemblyreaction was added to 30 μL chilled water along with 10 μL E cloni®(Lucigen, Middleton, Wis.) supreme competent cells. An aliquot of thetransformed cells were spot plated to check the transformationefficiency, where >100× coverage was required to continue. Thetransformed E cloni® cells were outgrown in 25 mL SOB+100 μg/mLcarbenicillin (carb). Glycerol stocks were generated from the saturatedculture by adding 500 μL 50% glycerol to 1000 μL saturated overnightculture. The stocks were frozen at −80° C. This step is optional,providing a ready stock of the cloned editing library. Alternatively,Gibson or another assembly of the editing cassettes and the vectorbackbone can be performed before each editing experiment. In thisexperiment, a 60-member triple editing plasmid pool was used, where eachediting plasmid targeted three genes: xylA, lacZ and galK.

Example V: Standard Plating Protocol

This protocol describes a standard plating protocol for nucleicacid-guided nuclease editing of bacterial cells. Materials: Outside ofstandard molecular biology tools, the following will be necessary:

TABLE 1 Product Vendor SOB Teknova LB Teknova LB agar plate with Teknovachloramphenicol/carbenicillin and 1% arabinose

Protocol: Immediately after transformation with the editing plasmidlibrary, the cell/DNA mixture was transferred to a culture tubecontaining 2.7 mL of SOB medium. Preparing 2.7 mL aliquots in 14 mLculture tubes prior to electroporation allowed for a faster recovery ofcells; the final volume of the recovery was 3 mL. All culture tubes wereplaced into a shaking incubator set to 250 RPM and 30° C. for threehours. While the cultures were recovering, the necessary number of LBagar plates with chloramphenicol and carbenicillin+1% arabinose wereremoved from the refrigerator and warmed to room temperature. Multipledilutions were used for each plating so as to have countable andisolated colonies on the plates. Plating suggestions:

TABLE 2 Sequencing type Dilution(s) suggested Volume to plate SinglePlex10⁻¹ through 10⁻³ 300 uL Amplicon None 300 uL (= 1/10^(th) recovery)

After three hours, the culture tubes were removed from the shakingincubator. First, plating for amplicon sequencing was performed byfollowing the above table. Plating beads were used to evenly distributethe culture over the agar. The beads were removed from the plate theplate was allowed to dry uncovered in a flow hood. While the plates weredrying, the remaining culture was used to perform serial dilutions,where the standard dilutions were 50 μL of culture into 450 uL ofsterile, 0.8% NaCl. The plate/tubes used for these dilutions (as well asthe original culture) were maintained at 4° C. in case additionaldilutions were needed to be performed based on colony counts. Platingfor whole genome sequencing was performed according to the Table 2.Additional or fewer dilutions may be used based on library/competentcell knowledge. The cultures were evenly spread across the agar usingsterile, plating beads. The beads were then removed from the plate andthe plates were allowed to dry uncovered in the flow hood. The plateswere incubated at 30° C. for 18 hours then the agar plates were removedfrom the incubator. If editing has been successful there will be aphenotypic readout on MacConkey agar.

The results for the p197 and p197-recA engine plasmids are shown inFIGS. 7A and 7B. Note in FIG. 7A the p197-recA engine plasmid reducedpost-plating CFUs by almost 2-orders of magnitude; however, 15/20colonies that grew were triple editors (see FIG. 7B). That is, if thecells did survive, 75% were triple editors.

The results for survival and percent editing for the p197, p197-recA,and p197-srpRrecA engine plasmids are shown in FIG. 8A-8C. FIG. 8A showsthe CFUs obtained for E coli transformed with the p197 engine vector,the p197-recA engine vector, and the p197-srpRrecA engine vector atuptake, at plating, and post-plating. Note that the post-plating CFU forthe p197-srpRrecA engine plasmid was comparable to that obtained withp197; however, the p197-recA engine plasmid had a low post-plating CFU.FIG. 8B shows the editing CFU and percentage of triple simultaneousedits for E. coli cells transformed with the p197 engine vector, thep197-recA engine vector and the p197-srpRrecA engine vector. Note thatalthough the overall editing rate for the p197-srpRrecA engine vectorwas lower than the p-197-recA engine vector, the rate for edited CFU ishigher. FIG. 8C is a table reporting the post-plating survival rate andthe percentage editing rate for E. coli cells reported in the graph inFIG. 8B. Note that the edit percentage for the p197 engine vector isapproximately 5%, the edit percentage for the p197-recA engine vector isapproximately 75%, and the edit percentage for the p197-srpRrecA enginevector is approximately 42%.

Example IV: Fully-Automated Singleplex RGN-Directed Editing Run

Singleplex automated genomic editing using MAD7 nuclease wassuccessfully performed with an automated multi-module instrument of thedisclosure. For examples of multi-module cell editing instruments, seeU.S. Pat. No. 10,253,316, issued 9 Apr. 2019; U.S. Pat. No. 10,329,559,issued 25 Jun. 2019; and U.S. Pat. No. 10,323,242, issued 18 Jun. 2019;and U.S. Ser. No. 16/412,175, filed 14 May 2019; Ser. No. 16/412,195,filed 14 May 2019; and Ser. No. 16/423,289, filed 28 May 2019, all ofwhich are herein incorporated by reference in their entirety.

An ampR plasmid backbone and a lacZ_F172* editing cassette wereassembled via Gibson Assembly® into an “editing vector” in an isothermalnucleic acid assembly module included in the automated instrument.lacZ_F172 functionally knocks out the lacZ gene. “lacZ_F172*” indicatesthat the edit happens at the 172nd residue in the lacZ amino acidsequence. Following assembly, the product was de-salted in theisothermal nucleic acid assembly module using AMPure beads, washed with80% ethanol, and eluted in buffer. The assembled editing vector andrecombineering-ready, electrocompetent E. Coli cells were transferredinto a transformation module for electroporation. The cells and nucleicacids were combined and allowed to mix for 1 minute, and electroporationwas performed for 30 seconds. The parameters for the poring pulse were:voltage, 2400 V; length, 5 ms; interval, 50 ms; number of pulses, 1;polarity, +. The parameters for the transfer pulses were: Voltage, 150V; length, 50 ms; interval, 50 ms; number of pulses, 20; polarity, +/−.Following electroporation, the cells were transferred to a recoverymodule (another growth module) and allowed to recover in SOC mediumcontaining chloramphenicol. Carbenicillin was added to the medium after1 hour, and the cells were allowed to recover for another 2 hours. Afterrecovery, the cells were held at 4° C. until recovered by the user.

After the automated process and recovery, an aliquot of cells was platedon MacConkey agar base supplemented with lactose (as the sugarsubstrate), chloramphenicol and carbenicillin and grown until coloniesappeared. White colonies represented functionally edited cells, purplecolonies represented un-edited cells. All liquid transfers wereperformed by the automated liquid handling device of the automatedmulti-module cell processing instrument.

The result of the automated processing was that approximately 1.0E⁻⁰³total cells were transformed (comparable to conventional benchtopresults), and the editing efficiency was 83.5%. The lacZ_172 edit in thewhite colonies was confirmed by sequencing of the edited region of thegenome of the cells. Further, steps of the automated cell processingwere observed remotely by webcam and text messages were sent to updatethe status of the automated processing procedure.

Example V: Fully-Automated Recursive Editing Run

Recursive editing was successfully achieved using the automatedmulti-module cell processing system. An ampR plasmid backbone and alacZ_V10* editing cassette were assembled via Gibson Assembly® into an“editing vector” in an isothermal nucleic acid assembly module includedin the automated system. Similar to the lacZ_F172 edit, the lacZ_V10edit functionally knocks out the lacZ gene. “lacZ_V10” indicates thatthe edit happens at amino acid position 10 in the lacZ amino acidsequence. Following assembly, the product was de-salted in theisothermal nucleic acid assembly module using AMPure beads, washed with80% ethanol, and eluted in buffer. The first assembled editing vectorand the recombineering-ready electrocompetent E. Coli cells weretransferred into a transformation module for electroporation. The cellsand nucleic acids were combined and allowed to mix for 1 minute, andelectroporation was performed for 30 seconds. The parameters for theporing pulse were: voltage, 2400 V; length, 5 ms; interval, 50 ms;number of pulses, 1; polarity, +. The parameters for the transfer pulseswere: Voltage, 150 V; length, 50 ms; interval, 50 ms; number of pulses,20; polarity, +/−. Following electroporation, the cells were transferredto a recovery module (another growth module) allowed to recover in SOCmedium containing chloramphenicol. Carbenicillin was added to the mediumafter 1 hour, and the cells were grown for another 2 hours. The cellswere then transferred to a centrifuge module and a media exchange wasthen performed. Cells were resuspended in TB containing chloramphenicoland carbenicillin where the cells were grown to OD600 of 2.7, thenconcentrated and rendered electrocompetent.

During cell growth, a second editing vector was prepared in anisothermal nucleic acid assembly module. The second editing vectorcomprised a kanamycin resistance gene, and the editing cassettecomprised a galK Y145* edit. If successful, the galK Y145* edit conferson the cells the ability to uptake and metabolize galactose. The editgenerated by the galK Y154* cassette introduces a stop codon at the154th amino acid reside, changing the tyrosine amino acid to a stopcodon. This edit makes the galK gene product nonfunctional and inhibitsthe cells from being able to metabolize galactose. Following assembly,the second editing vector product was de-salted in the isothermalnucleic acid assembly module using AMPure beads, washed with 80%ethanol, and eluted in buffer. The assembled second editing vector andthe electrocompetent E. Coli cells (that were transformed with andselected for the first editing vector) were transferred into atransformation module for electroporation, using the same parameters asdetailed above. Following electroporation, the cells were transferred toa recovery module (another growth module), allowed to recover in SOCmedium containing carbenicillin. After recovery, the cells were held at4° C. until retrieved, after which an aliquot of cells were plated on LBagar supplemented with chloramphenicol, and kanamycin. To quantify bothlacZ and galK edits, replica patch plates were generated on two mediatypes: 1) MacConkey agar base supplemented with lactose (as the sugarsubstrate), chloramphenicol, and kanamycin, and 2) MacConkey agar basesupplemented with galactose (as the sugar substrate), chloramphenicol,and kanamycin. All liquid transfers were performed by the automatedliquid handling device of the automated multi-module cell processingsystem.

In this recursive editing experiment, 41% of the colonies screened hadboth the lacZ and galK edits, the results of which were comparable tothe double editing efficiencies obtained using a “benchtop” or manualapproach.

While this invention is satisfied by embodiments in many differentforms, as described in detail in connection with preferred embodimentsof the invention, it is understood that the present disclosure is to beconsidered as exemplary of the principles of the invention and is notintended to limit the invention to the specific embodiments illustratedand described herein. Numerous variations may be made by persons skilledin the art without departure from the spirit of the invention. The scopeof the invention will be measured by the appended claims and theirequivalents. The abstract and the title are not to be construed aslimiting the scope of the present invention, as their purpose is toenable the appropriate authorities, as well as the general public, toquickly determine the general nature of the invention. In the claimsthat follow, unless the term “means” is used, none of the features orelements recited therein should be construed as means-plus-functionlimitations pursuant to 35 U.S.C. § 112, ¶6.

We claim:
 1. A method for increasing observed editing in a multiplexedCRISPR nuclease editing system in bacteria comprising: a) providingelectrocompetent bacteria cells; b) providing: i) an engine vectorcomprising: a first inducible promoter driving expression of a codingsequence for a CRISPR nuclease; a bacterial origin of replication; asecond inducible promoter or a constitutive promoter driving expressionof a coding sequence for a recA protein; and a selection marker; and ii)an editing vector comprising: the second inducible promoter or theconstitutive promoter driving transcription of at least three editingcassettes where each editing cassette comprises a gRNA sequence and adonor DNA sequence to be transcribed; a bacterial origin of replication;and a selection marker; c) transforming the electrocompetent bacteriacells with the engine and editing vectors; d) allowing transcription ofthe recA protein and the at least three editing cassettes from thesecond inducible promoter or the constitutive promoter; e) followingtranscription of the recA protein and the at least three editingcassettes, inducing transcription of the CRISPR nuclease; f) allowingthe transformed cells to edit; and g) pooling the edited cells orselecting small colonies of edited cells.
 2. The method of claim 1,wherein the nuclease is MAD7.
 3. The method of claim 1, wherein thenuclease is Cas9.
 4. The method of claim 1, wherein the coding sequencefor the recA protein is a coding sequence for a recA fusion protein. 5.The method of claim 4, wherein the recA fusion protein is a recA-srpRfusion protein.
 6. The method of claim 5, wherein the recA-srpR fusionprotein comprises an in-frame fusion protein comprising a codingsequence of the srpR protein at an N-terminal portion of the in-framefusion protein and the coding sequence for the recA protein codingsequence at a C-terminal portion of the in-frame fusion protein.
 7. Themethod of claim 1, wherein the engine vector comprises a coding sequencefor c1857 and the first inducible promoter is a pL promoter drivingexpression of the nuclease.
 8. The method of claim 1, wherein the enginevector further comprises coding sequences for a λRed recombineeringsystem.
 9. The method of claim 1, wherein the second inducible promoterdriving transcription of the at least three editing cassettes is a pLinducible promoter.
 10. The method of claim 9, wherein the editingvector comprises the second inducible promoter driving transcription ofat least four editing cassettes.
 11. The method of claim 1, wherein theselection marker on the engine vector and the selection marker on theediting vector are different selection markers.
 12. The method of claim1, further comprising the steps of, after the pooling or selecting step:h) making the edited bacteria cells electrocompetent; i) providing asecond editing vector comprising the second inducible promoter or theconstitutive promoter driving transcription of at least three editingcassettes where each editing cassette comprises a gRNA sequence and adonor DNA sequence to be transcribed; a bacterial origin of replication;and a selection marker; j) transforming the electrocompetent bacteriacells with the second editing vector; k) allowing transcription of therecA protein and the at least three editing cassettes from the secondinducible promoter or the constitutive promoter; l) followingtranscription of the recA protein and the at least three editingcassettes, inducing transcription of the CRISPR nuclease; m) allowingthe transformed cells to edit; and n) pooling the twice-edited cells.13. A method for increasing observed editing in a multiplexed CRISPRnuclease editing system in bacteria comprising: a) providingelectrocompetent bacteria cells; b) providing: c) an engine vectorcomprising: i. a first inducible promoter driving expression of a codingsequence for a CRISPR nuclease; ii. a bacterial origin of replication;iii. a second inducible promoter or a constitutive promoter drivingexpression of a coding sequence for a recA protein; iv. a λRedrecombineering system; and v. a first selection marker; d) an editingvector comprising: i. a second inducible promoter or a constitutivepromoter driving transcription of at least three editing cassettes whereeach editing cassette comprises a gRNA sequence and a donor DNA sequenceto be transcribed; ii. a bacterial origin of replication; and iii. asecond selection marker; e) transforming the electrocompetent bacteriacells with the engine and editing vectors; f) allowing transcription ofthe recA protein and the at least three editing cassettes from thesecond inducible promoter or the constitutive promoter; g) followingtranscription of the recA protein and the at least three editingcassettes, inducing transcription of the CRISPR nuclease; h) allowingthe transformed cells to edit; and i) pooling the edited cells orselecting small colonies of edited cells.
 14. The method of claim 13,wherein the nuclease is MAD7.
 15. The method of claim 13, wherein thenuclease is Cas9.
 16. The method of claim 13, wherein the codingsequence for the recA protein is a coding sequence for a recA fusionprotein.
 17. The method of claim 16, wherein the recA fusion protein isa recA-srpR fusion protein.
 18. The method of claim 17, wherein therecA-srpR fusion protein comprises an in-frame fusion protein comprisinga coding sequence of the srpR protein at an N-terminal portion of thein-frame fusion protein and the coding sequence for the recA proteincoding sequence at a C-terminal portion of the in-frame fusion protein.19. The method of claim 13, wherein the engine vector comprises a codingsequence for c1857 and an inducible pL promoter drives expression of thenuclease and the at least three editing cassettes.
 20. The method ofclaim 13, wherein the editing vector comprises a second induciblepromoter driving transcription of at least four editing cassettes.